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Role of Ceramides in Skin Stress: Ultraviolet Light, Tape Stripping and Crowding
Abstract
Stratum corneum intercellular lipids regulate skin water barrier function and water-holding capacity; their modification may impair these properties. Physical and chemical stresses diminish barrier function. Acute barrier disruption by tape stripping increases sphingomyelinase and serine palmitoyltransferase activity; ceramide contents are increased to restore barrier function. Overcrowding stress induces dry skin, and the barrier function impairment correlates with decreased skin cera- mides. The effect of UV irradiation on ceramide content and barrier function varies with doses and UV wavelength. Stress-induced ceramide generation may induce apoptosis in cultured human keratinocytes and restore barrier function. This review focuses on the role of ceramides in physical and chemical stress, suggesting that refinement and extension of this academic domain may lead to therapeutic advances.
... A family of ceramides and phosphorylcholine can be generated by the activity of sphingomyelinase from glucosylceramide and sphingomyelin precursors (Jensen et al., 2005). The action of this enzyme is critical for the skin barrier function (Choi and Maibach, 2003). Bacteria, yeast, and mammalian cells produce sphingomyelinase. ...
Age and diabetes related slow-healing or chronic wounds may result in morbidity and mortality through persistent biofilms infections and prolonged inflammatory phase. Nano-materials [metal/metal oxide NPs (39%), lipid vehicles (21%), polymer NPs (19%), ceramic nanoparticles (NPs) (14%), and carbon nanomaterials (NMs) (7%)] can be introduced as a possible next-generation therapy because of either their intrinsic wound healing activity or via carrying bioactive compounds including, antibiotics, antioxidants, growth factor or stem cell. The nanomaterials have been shown to implicate in all four stages of wound healing including hemostasis (polymer NPs, ceramic NPs, nanoceria-6.1%), inflammation (liposome/vesicles/solid lipid NPs/polymer NPs/ceramic NPs/silver NPs/gold NPs/nanoceria/fullerenes/carbon-based NPs-32.7%), proliferation (vesicles/liposome/solid lipid NPs/gold NPs/silver NPs/iron oxide NPs/ceramic NPs/copper NPs/self-assembling elastin-like NPs/nanoceria/micelle/dendrimers/polymer NPs-57.1%), remodeling (iron oxide NPs/nanoceria-4.1%). Natural compounds from alkaloids, flavonoids, retinoids, volatile oil, terpenes, carotenoids, or polyphenolic compounds with proven antioxidant, anti-inflammatory, immunomodulatory, or antimicrobial characteristics are also well known for their potential to accelerate the wound healing process. In the current paper, we survey the potential and properties of nanomaterials and microbial compounds in improving the process of wound and scar healing. Finally, we review the potential biocompounds for incorporation to nano-material in perspective to designate more effective or multivalent wound healing natural or nano-based drugs.
... Further studies investigating the epidermal growth kinetic after standardized injury of healthy [100][101][102] and atopic dermatitis skin [103] by tape stripping were performed. Recently, Choi et al. showed that tape stripping induces the production of cytokines such as interleukins, tumor necrosis factor α, and γ-interferon [104], enhances the enzymatic activity, and increases the ceramides synthesis [105]. The artificial removal of the stratum corneum barrier by tape stripping has become a frequently used model for simulating diseased skin [106], and for assessing the efficacy of skin care products in restoring the barrier [107,108]. ...
... While mammalian neutral SMase are membrane-bound proteins, bacterial SMase are secretory proteins released from cells into the media [6]. Sphingomyelinase activity has been demonstrated to be important for skin barrier function as ceramides generated by SMase are major components of the intercellular lipids of the stratum corneum [7]. A decrease of ceramide in the stratum corneum causes water loss and barrier dysfunction in the epidermis, including a loss of protection against antigens and bacteria [8]. ...
The present research aims to optimize the sphingomyelinase (SMase) activity produced by Lactobacillus rhamnosus FTDC 8313 using divalent metal ions via response surface methodology and to further study the effects of the divalent metal ions on SMase activity using molecular modeling approach. This study also aimed to assess the possibility of increasing ceramide levels in vitro on cultured keratinocytes upon treatment with the extracellular extract of the optimized L. rhamnosus FTDC 8313. Using a central composite design, an optimum point of SMase activity (6.54 mU ml−1) was produced from a combination of 0.65% (w/v) MnSO4 and 0.82% (w/v) MgSO4. 3D response surface indicated that the altered availability of the two ions (Mn2+ and Mg2+) reduced their effects on SMase activity. In addition, the treatment of the HaCaT cells with optimized extracellular extract of L. rhamnosus FTDC8313 significantly increased (P < 0.05) the conversion of sphingomyelin to ceramide as compared to the control. Molecular docking demonstrated that the addition of Mn2+ and Mg2+ into the active site of SMase improved the binding affinity between the SMase and sphingomyelin based on its free energy of binding as well as the interaction distances between the important catalytic residues Glu53 and His296.
... Sphingomyelinase (SMase) is an enzyme that generates a family of ceramides and phosphorylcholine from glucosylceramide and sphingomyelin precursors for the development of extracellular lipid bilayers in the stratum corneum (Jensen et al. 2005). SMase activity has been demonstrated to be important for skin barrier function (Choi and Maibach 2003). A decrease in ceramide in the stratum corneum causes water loss and barrier dysfunction in the epidermis, including a loss of protection against antigens and bacteria (Mizutani et al. 2009). ...
Probiotics have been extensively reviewed for decades, emphasizing on improving general gut health. Recently, more studies showed that probiotics may exert other health promoting effects beyond gut well-being, attributed to the rise of the gut-brain-axis correlations. Some of these new benefits include skin health such as improving atopic eczema, atopic dermatitis, healing of burn and scars, skin rejuvenating properties, and improving skin innate immunity. Increasing evidences have also showed that bacterial compounds such as cell wall fragments, their metabolites and dead bacteria can elicit certain immune responses on the skin and improve skin barrier functions. This review aimed to underline the mechanisms or the exact compounds underlying the benefits of bacterial extract on the skin based on evidences from in vivo and in vitro studies. This review could be of help in screening of probiotic strains with potential dermal enhancing properties for topical applications. © 2013 The Authors Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.
Skin is a multi-stratified, multi-compartmental membrane with complex structural features that represent on evolutionary compromise between a need for mechanical protection and those of sensing the environment and exchanging materials and energy with it. Amniote vertebrates evolved a skin that resists water loss and physical abrasion while simultaneously allowing for changes of shape and movement. The epidermis found in the majority of terrestrial vertebrates includes a multi-layered barrier of keratin and lamellar lipids that protect the internal cellular environment from the dehydrating effects of surrounding air. The compositional features of these lipids together with a stratified keratogenic matrix are key to the effectiveness of the water barrier function. In mammals, molar ratios of ceramides, cholesterol and free fatty acids are critical to barrier function and are derived enzymatically from lamellar body organelles whose contents are exocytosed from keratinocytes into the extracellular spaces. The hydration status and flexibility of the epidermis depend on hygroscopic proteins, osmotically active elements, and the integrity of the lipid water barrier. Competence of the barrier is regulated in response to environment or traumatic injury to skin, and the water flux or content of stratum corneum, environmental humidity, calcium gradients, pH, altered osmotic pressure, and induction of cytokines and filaggrin proteolytic pathways are involved in complex, interactive ways. The resistance of skin to evaporative water loss is heritable, phenotypically plastic, and subject to natural selection in perhaps numerous species. Both model systems and comparative evolutionary studies provide important insights and are important for future advances in understanding skin function. Future knowledge from such research will have application to a variety of issues in dermatology and cosmetic medicine.
Ceramides are the major component of the extracellular lipids that comprise the epidermal permeability barrier. They are derived
from glucosylceramides (GlcCer) upon their extrusion from lamellar granules into the extracellular space in the upper layers
of the epidermis. To better understand the regulation of the unique pathway for ceramide production in epidermis, we have
studied the activity of the enzyme responsible for GlcCer synthesis, ceramide glucosyltransferase (CerGlc transferase), during
keratinocyte culture differentiation. Human keratinocyte cultures were expanded in low calcium keratinocyte growth medium
(KGM) and then switched to either normal calcium KGM (nKGM) or “complete” Dulbecco's modified Eagle's medium/Ham's F-12 (3:1)
supplemented with 10% fetal bovine serum (cDMEM). At 7 and 10 days after the medium switch, electron microscopy revealed that
cDMEM cultures were more fully differentiated morphologically and contained numerous lamellar granules. The GlcCer/DNA content
of cDMEM cultures increased to 6 times that of day 0 cultures and was nearly 4 times greater than that of nKGM cultures, whereas
the total lipid/DNA content of cDMEM cultures increased to only 1.8 times that of day 0 cultures and was ∼1.2 times that of
nKGM cultures. CerGlc transferase activity/DNA increased 6 times in cDMEM cultures but <1.5 times in nKGM cultures. By contrast,
β-glucocerebrosidase activity, which is responsible for the conversion of GlcCer to ceramide, increased to a similar extent
in both differentiating culture systems. Treatment of cultures with the reversible CerGlc transferase inhibitor, DL-threo-1-phenyl-2-(palmitoylamino)-3-morpholino-1-propanol, prevented the increase of GlcCer in cDMEM cultures, and blocked conversion
of exogenously added ceramide to GlcCer. A low level of CerGlc transferase activity, relative to that in differentiated keratinocytes,
was detected in cultures of other human cell types. These results indicate that CerGlc transferase activity is induced during
epidermal differentiation and that regulation of this enzyme may be an important determinant of the specialized production
and compartmentalization of epidermal sphingolipids.
Epidermal TNF expression increases in response to cutaneous permeability barrier disruption and wound healing. TNF signaling is mediated by acid and neutral sphingomyelinases (A- and N-SMase), which generate ceramide, an important regulator of proliferation, differentiation, and apoptosis. In the epidermis, ceramide is known to be an integral part of the extracellular stratum corneum (SC) lipid bilayers that constitute the permeability barrier of the skin. We show here that topical application of TNF after experimental injury to the SC of hairless mice (hr(-/-)) enhances barrier repair. In TNF receptor p55-deficient (TNF-R55-deficient) mice (hr(+/+)), cutaneous barrier repair was delayed compared with wild-type (hr(+/+)) or TNF-R75-deficient (hr(+/+)) animals. After barrier disruption in hairless (hr(-/-)) and wild-type (hr(+/+)), but not in TNF-R55-deficient (hr(+/+)) mice, the enzymatic activities of both A-SMase and N-SMase were significantly enhanced. Stimulation of SMase activities was accompanied by an increase in C(24)-ceramide levels. Most A-SMase activity in hairless mice (hr(-/-)) was found in the outer epidermal cell layers and colocalized in the lamellar bodies with A-SMase and sphingomyelin. Reduction of epidermal A-SMase activity by the inhibitor imipramine resulted in delayed permeability barrier repair after SC injury. Together, these results suggest that TNF-R55 signaling pathways contribute to cutaneous permeability barrier repair through SMase-mediated generation of ceramide.
Ceramides, which derive from the hydrolysis of glucosylceramide (GlcCer), are the predominant lipid species in the stratum corneum and are critical for epidermal permeability barrier homeostasis. UDP-glucose:ceramide glucosyltransferase (GlcCer synthase) (EC 2.4.1.80) catalyzes the glucosylation of ceramide to form GlcCer. Recently, we demonstrated a progressive increase in GlcCer synthase expression during fetal barrier development, while others have reported increased GlcCer synthase activity with differentiation of cultured human keratinocytes. To further delineate the role of GlcCer synthase in barrier homeostasis, we determined GlcCer synthase activity and localization in hairless mouse epidermis, both under basal conditions and after acute barrier perturbation. Under basal conditions, GlcCer synthase activity localizes predominantly (approximately 80%) to the dithiothreitol-separated outer epidermis; i.e., 6.2+/-0.6 versus 1.2+/-0.1 pmol/min/mg for outer vs. lower epidermis, respectively (P < 0.0001). Although acute barrier disruption does not up-regulate epidermal GlcCer synthase activity at any time point up to 24 h, GlcCer synthase is required for barrier homeostasis: topical d,1-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (P4), a specific GlcCer synthase inhibitor, applied immediately after acute barrier disruption, causes a delay in barrier recovery attributable to specific enzyme inhibition. These findings demonstrate first, that GlcCer synthase activity predominates in the outer epidermis, consistent with an increased formation of GlcCer during barrier ontogenesis and maintenance. Second, GlcCer synthase activity is required for normal permeability barrier homeostasis. Third, baseline epidermal GlcCer synthase activity appears to accommodate acute challenges to the barrier.
Stratum corneum lipids comprise an approximately equimolar mixture of sphingolipids, cholesterol, and free fatty acids, arranged as intercellular membrane bilayers that are presumed to mediate the epidermal permeability barrier. Prior studies have shown that alterations in epidermal barrier function lead to a rapid increase in cholesterol and fatty acid synthesis which parallels the early stages of the repair process. Despite an abundance of indirect evidence for their role in the barrier, the importance of sphingolipids has yet to be demonstrated directly. Whereas sphingolipid synthesis also increases during barrier repair, this response is delayed in comparison to cholesterol and fatty acid synthesis (Holleran, W.M., et al. 1991. J. Lipid Res. 32:1151-1158). To further delineate the role of sphingolipids in barrier homeostasis, we assessed the impact of inhibition of sphingolipid synthesis on epidermal barrier recovery. A single topical application of beta-chloro-L-alanine (beta-CA), an irreversible inhibitor of serine-palmitoyl transferase (SPT), applied to acetone-treated skin of hairless mice resulted in: (a) greater than 75% inhibition of SPT activity at 30 min (P less than 0.001); (b) a global decrease in sphingolipid synthesis between 1 and 3 h (P less than 0.02); (c) reduction of epidermal sphingolipid content at 18 h (P less than 0.01); (d) delayed reaccumulation of histochemical staining for sphingolipids in the stratum corneum; and (e) reduced numbers and contents of lamellar bodies in the stratum granulosum. Finally, despite its immediate, marked diminution of sphingolipid synthesis, beta-CA slowed barrier recovery only at late time points (greater than 6 h) after acetone treatment. This inhibition was overridden by coapplications of ceramides (the distal SPT product), indicating that the delay in repair was not due to non-specific toxicity. These studies demonstrate a distinctive role for epidermal sphingolipids in permeability barrier homeostasis.
Recent work concerning the interaction of UVA radiation (320-380 nm) with cultured cells is reviewed with particular emphasis on the involvement of cellular oxidative stress in the biological effects of this radiation on eucaryotic cells. Possible chromophores are considered and their role in generation of various oxidant species including hydrogen peroxide, superoxide anion, singlet oxygen and hydroxyl radical. DNA and membranes are discussed as possible targets for the lethal action of long wavelength radiation. Four mechanisms of cellular defence are proposed: (1) DNA repair; (2) antioxidant enzymes; (3) endogenous free radical quenchers; (4) inducible protection.
Aged epidermis displays altered drug permeability, increased susceptibility to irritant contact dermatitis, and often severe xerosis, suggesting compromise of the aged epidermal barrier. To delineate the functional, structural, and lipid biochemical basis of epidermal aging, we compared barrier function in young (20-30 yr) vs aged (> 80 yr) human subjects, and in a murine model. Baseline transepidermal water loss in both aged humans and senescent mice was subnormal. However, the aged barrier was perturbed more readily with either acetone or tape stripping (18 +/- 2 strippings vs 31 +/- 5 strippings in aged vs young human subjects, respectively). Moreover, after either acetone treatment or tape stripping, the barrier recovered more slowly in aged than in young human subjects (50 and 80% recovery at 24 and 72 h, respectively, in young subjects vs 15% recovery at 24 h in aged subjects), followed by a further delay over the next 6 d. Similar differences in barrier recovery were seen in senescent vs young mice. Although the total lipid content was decreased in the stratum corneum of aged mice (approximately 30%), the distribution of ceramides (including ceramide 1), cholesterol, and free fatty acids was unchanged. Moreover, a normal complement of esterified, very long-chain fatty acids was present. Finally, stratum corneum lamellar bilayers displayed normal substructure and dimensions, but were focally decreased in number, with decreased secretion of lamellar body contents. Thus, assessment of barrier function in aged epidermis under basal conditions is misleading, since both barrier integrity and barrier repair are markedly abnormal. These functional changes can be attributed to a global deficiency in all key stratum corneum lipids, resulting in decreased lamellar bilayers in the stratum corneum interstices. This constellation of findings may explain the increased susceptibility of intrinsically aged skin to exogenous and environmental insults.
The intercellular spaces of the outermost layers of the epidermis (stratum corneum, SC) of terrestrial mammals contain a mixture of lipids, enriched in ceramides that are critical for the epidermal permeability barrier. Whereas glucosylceramides (GlcCer) are synthesized in abundance in the epidermis, they disappear coincident with an increase of ceramides (Cer) in the SC. Hence, hydrolysis of GlcCer to Cer by beta-glucocerebrosidase (GlcCer'ase), may be required for permeability barrier homeostasis. We determined first whether modulations in epidermal GlcCer'ase activity and mRNA levels occur in response to barrier disruption; and second, how GlcCer'ase inhibitors influence barrier function and SC membrane ultrastructure. Barrier disruption significantly increased epidermal GlcCer'ase mRNA levels, with a 2.8-fold increase over untreated control levels at 8 h (P < 0.01). GlcCer'ase activity was increased in whole epidermis (34%; P < 0.02) 24 h after barrier disruption. Localization of GlcCer'ase activity showed an increase (33%; P < 0.05) in the outer epidermis (SC and stratum granulosum), without a change in lower epidermal activity (stratum spinosum and stratum basale). Furthermore, a single topical application of the GlcCer'ase inhibitor, bromoconduritol-B-epoxide (BrCBE), inhibited enzyme activity (98%) and significantly delayed permeability barrier recovery after acetone treatment. In addition, BrCBE treatment disrupted SC intercellular lamellar bilayers, without evidence of cellular toxicity. These results indicate that epidermal processing of GlcCer to Cer by GlcCer'ase is required for barrier homeostasis, and that this important enzymatic step is regulated by barrier requirements.
Hydrolysis of glucosylceramide by beta-glucocerebrosidase results in ceramide, a critical component of the intercellular lamellae that mediate the epidermal permeability barrier. A subset of type 2 Gaucher patients displays ichthyosiform skin abnormalities, as do transgenic Gaucher mice homozygous for a null allele. To investigate the relationship between glucocerebrosidase deficiency and epidermal permeability barrier function, we compared the stratum corneum (SC) ultrastructure, lipid content, and barrier function of Gaucher mice to carrier and normal mice, and to hairless mice treated topically with bromoconduritol B epoxide (BrCBE), an irreversible inhibitor of glucocerebrosidase. Both Gaucher mice and BrCBE-treated mice revealed abnormal, incompletely processed, lamellar body-derived sheets throughout the SC interstices, while transgenic carrier mice displayed normal bilayers. The SC of a severely affected type 2 Gaucher's disease infant revealed similarly abnormal ultrastructure. Furthermore, the Gaucher mice demonstrated markedly elevated transepidermal water loss (4.2 +/- 0.6 vs < 0.10 g/m2 per h). The electron-dense tracer, colloidal lanthanum, percolated between the incompletely processed lamellar body-derived sheets in the SC interstices of Gaucher mice only, demonstrating altered permeability barrier function. Gaucher and BrCBE-treated mice showed < 1% and < 5% of normal epidermal glucocerebrosidase activity, respectively, and the epidermis/SC of Gaucher mice demonstrated elevated glucosylceramide (5- to 10-fold), with diminished ceramide content. Thus, the skin changes observed in Gaucher mice and infants may result from the formation of incompetent intercellular lamellar bilayers due to a decreased hydrolysis of glucosylceramide to ceramide. Glucocerebrosidase therefore appears necessary for the generation of membranes of sufficient functional competence for epidermal barrier function.
The interstices of the mammalian stratum corneum contain lipids in a system of continuous membrane bilayers critical for the epidermal permeability barrier. During the transition from inner to outer stratum corneum, the content of polar lipids including glucosylceramides, decreases while ceramide content increases. We investigated whether inhibition of glucosylceramide hydrolysis would alter epidermal permeability barrier function. Daily topical applications of bromoconduritol B epoxide (BrCBE) to intact murine skin selectively inhibited beta-glucocerebrosidase, increased glucosylceramide content of stratum corneum with ceramide content remaining largely unchanged, and caused a progressive, reversible decrease in barrier function. Histochemistry of inhibitor-treated epidermis revealed persistence of periodic acid-Schiff-positive staining in stratum corneum cell membranes, consistent with retention of hexose moieties. Electron microscopy of inhibitor-treated samples revealed no evidence of toxicity or changes in the epidermal lipid delivery system. However, immature membrane structures persisted in the intercellular spaces throughout the stratum corneum, with reappearance of mature membrane structures progressing outward from the lower stratum corneum upon termination of BrCBE. Finally, the induced barrier abnormality was not reversed by coapplications of ceramide. These data demonstrate that glucosylceramide hydrolysis is important in the formation of the epidermal permeability barrier, and suggest that accumulation of glucosylceramides in stratum corneum intercellular membrane domains leads to abnormal barrier function.
Damage to skin collagen and elastin (extracellular matrix) is the hallmark of long-term exposure to solar ultraviolet irradiation, and is believed to be responsible for the wrinkled appearance of sun-exposed skin. We report here that matrix-degrading metalloproteinase messenger RNAs, proteins and activities are induced in human skin in vivo within hours of exposure to ultraviolet-B irradiation (UVB). Induction of metalloproteinase proteins and activities occurred at UVB doses well below those that cause skin reddening. Within minutes, low-dose UVB upregulated the transcription factors AP-1 and NF-kappa B, which are known to be stimulators of metalloproteinase genes. All-trans retinoic acid, which transrepresses AP-1 (ref. 8), applied before irradiation with UVB, substantially reduced AP-1 and metalloproteinase induction. We propose that elevated metalloproteinases, resulting from activation of AP-1 and NF-kappa B by low-dose solar irradiation, degrade collagen and elastin in skin. Such damage, if imperfectly repaired, would result in solar scars, which through accumulation from a lifetime of repeated low-dose sunlight exposure could cause premature skin ageing (photoageing).
Development of atherosclerosis is believed to involve proliferation of smooth muscle cells (SMC). Our laboratory previously demonstrated that the growth of bovine aortic SMC was stimulated by mildly oxidized low density lipoproteins (oxLDL) and that the mitogenic effect of oxLDL was greater than that induced by native LDL (Augé, N., Pieraggi, M. T., Thiers, J. C., Nègre-Salvayre, A., and Salvayre R.(1995) Biochem. J. 309, 1015-1020). Since the lipid mediator ceramide has been described to be proliferative, the present work aimed at studying the potential involvement of the so-called sphingomyelin-ceramide pathway in the signal transduction cascade induced by oxLDL. Incubation of SMC with UV-oxidized LDL induced sphingomyelin hydrolysis (32%), which peaked at 60 min and was accompanied by a concomitant increase of intracellular ceramide level. The effect of oxidized LDL on sphingomyelin turnover exhibited the same LDL dose dependence as their mitogenic effect. Exogenous bacterial sphingomyelinase induced sphingomyelin hydrolysis and ceramide generation and also stimulated cell growth, in contrast to exogenous phospholipases A2, C, or D. This mitogenic effect was reproduced by incubating the cells with the cell-permeant ceramides, N-acetyl- and N-hexanoylsphingosines. Altogether, these data strongly suggest for the first time that activation of the sphingomyelin-ceramide pathway may play a pivotal role in the oxLDL-induced SMC proliferation and atherogenesis.
Glucosylceramides, delivered to the stratum corneum interstices by exocytosis of lamellar body contents, are enzymatically hydrolyzed to ceramides, which are major components of the lipid lamellar bilayers that mediate epidermal barrier function. Because this conversion is critical for permeability barrier homeostasis in the adult animal, in this study we measured the changes in activities of the enzymes responsible for the synthesis of glucosylceramide and its conversion to ceramide, UDP-glucose:ceramide glucosyltransferase (GC synthase) and beta-glucocerebrosidase (beta-GlcCer'ase), respectively, during fetal barrier formation. In epidermis from rats of gestational age 17-21 days, GC synthase activity peaked on day 19, prior to barrier competence, whereas beta-GlcCer'ase activity rose throughout barrier formation, exhibiting a 5-fold increase over this time period. beta-GlcCer'ase protein rose in parallel with activity, as did mRNA levels. Enzyme activities in skin explants from 17-day fetal rats, incubated up to 4 days in hormone- and serum-free media, paralleled those measured at corresponding time points in utero. Incubation with hormones that accelerate barrier development had minimal effects on GC synthase activity, whereas beta-GlcCer'ase activity was significantly increased after 1 or 2 days in culture. Finally, inhibition of beta-GlcCer'ase with conduritol B epoxide prevented barrier development in vitro and was accompanied by abnormalities in the lamellar bilayer ultrastructure of the stratum corneum. These data indicate that both synthesis and hydrolysis of glucosylceramide are regulated during fetal development. Furthermore, the enzymatic hydrolysis of glucosylceramide to ceramide is essential for fetal barrier ontogenesis.
An important component of barrier function in human epidermis is contributed by ceramides that are bound by ester linkages to undefined proteins of the cornified cell envelope (CE). In this paper, we have examined the protein targets for the ceramide attachment. By partial saponification of isolated foreskin epidermal CEs followed by limited proteolysis, we have recovered several lipopeptides. Biochemical and mass spectroscopic characterization revealed that all contained near stoichiometric amounts of ceramides of masses ranging from about 690 to 890 atomic mass units, of which six quantitatively major species were common. The array of ceramides was similar to that obtained from pig skin, the composition of which is known, thereby providing strong indirect data for their fatty acid and sphingosine compositions. The recovered peptides accounted for about 20% of the total foreskin CE ceramides. By amino acid sequencing, about 35% of the peptides were derived from ancestral glutamine-glutamate-rich regions of involucrin, an important CE structural protein. Another 18% derived from rod domain sequences of periplakin and envoplakin, which are also known or suspected CE proteins. Other peptides were too short for unequivocal identification. Together, these data indicate that involucrin, envoplakin, periplakin, and possibly other structural proteins serve as substrates for the attachment of ceramides by ester linkages to the CE for barrier function in human epidermis.
The enzyme serine palmitoyltransferase (SPT; EC 2.3.1.50), which catalyzes the first committed and rate-limiting step in sphingolipid synthesis, is up-regulated in the epidermis as part of the homeostatic repair in response to permeability barrier perturbation. Moreover, UVB exposure, which also perturbs the barrier, up-regulates sphingolipid synthesis, but the basis for this increase is not known. The recent isolation of cDNAs for SPT (i.e., LCB1 and LCB2) allow molecular regulation studies to be performed. Therefore, we determined whether UVB exposure alters mRNA, protein, or activity levels for SPT in cultured human keratinocytes (CHKs) as a mechanism for regulation of epidermal sphingolipid synthesis. In CHK, transcripts for both LCB1 (3.0 kb) and LCB2 (2.3 kb) are evident by Northern blot analysis, and UVB exposure (23 mJ/cm²) induces a delayed 1.8 to 3.3-fold increase in LCB2 mRNA levels (P < 0.01) 48 h after treatment versus non-irradiated control cells. In contrast, neither LCB1 nor a second LCB2 transcript (8.0 kb) changed significantly. Likewise, Lcb2 protein levels (by Western blot analysis), as well as SPT activity, increase in parallel with the increased LCB2 mRNA. Finally, incorporation of [¹⁴C]-acetate into sphingolipids was increased significantly 48 h after UVB treatment. Together, these results demonstrate that CHKs respond to UVB by increasing sphingolipid synthesis, primarily through increases in both LCB2 mRNA and protein levels, leading to increased SPT activity. These results demonstrate one mechanism (UVB) whereby SPT is regulated at the molecular level, and suggest further that epidermis up-regulates sphingolipid synthesis at both the mRNA and protein levels in response to UVB.
—Farrell, A. M., Y. Uchida, M. M. Nagiec, I. R. Harris, R. C. Dickson, P. M. Elias, and W. M. Holleran. UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes. J. Lipid Res. 1998. 39: 2031–2038.
We investigated xylene and toluene-induced skin irritation following a 24-h application to the skin of human subjects. Irritation was monitored by non-invasive methods: colorimetry, transepidermal water loss (TEWL), and visual scoring. The profile and composition of seven major skin lipids following skin irritation was determined. These data were used to define parameters for the identification of high risk subjects. The lipids in stratum corneum samples obtained from human volunteers (solvent-treated and untreated sites) were extracted, analyzed by thin layer chromatography (TLC) and quantified by video-densitometry. Following their exposure to xylene or toluene, all subjects developed some irritation, while four of them had extensive reactions (bullae). Visual score and colorimetry measurements exhibited high responses at all the observation times while TEWL showed an increase only at the 24-h control. Basal lipid composition of subjects with the greatest irritation had a significantly lower level of ceramides compared to the less reactive subjects (p < 0.05). The potential of organic solvents to act as skin irritants can be estimated and defined by these noninvasive methods. Analysis of baseline lipid skin composition may be a predictive test to identify heightened sensitivity to certain irritants.
There are several skin diseases in which the lipid composition in the intercellular matrix of the stratum corneum is different from that of healthy human skin. It has been shown that patients suffering from atopic dermatitis have a reduced ceramide content in the stratum corneum, whereas in the stratum corneum of lamellar ichthyosis patients, the amount of free fatty acids is decreased and the ceramide profile is altered. Both patient groups also show elevated levels of transepidermal water loss indicative of an impaired barrier function. As ceramides and free fatty acids are essential for a proper barrier function, we hypothesized that changes in the composition of these lipids would be reflected in the lipid organization in stratum corneum of atopic dermatitis and lamellar ichthyosis patients. We investigated the lateral lipid packing using electron diffraction and the lamellar organization using freeze fracture electron microscopy. In atopic dermatitis stratum corneum, we found that, in comparison with healthy stratum corneum, the presence of the hexagonal lattice (gel phase) is increased with respect to the orthorhombic packing (crystalline phase). In lamellar ichthyosis stratum corneum, the hexagonal packing was predominantly present, whereas the orthorhombic packing was observed only occasionally. This is in good agreement with studies on stratum corneum lipid models that show that the presence of long-chain free fatty acids is involved in the formation of the orthorhombic packing. The results of this study also suggest that the ceramide composition is important for the lateral lipid packing. Finally, using freeze fracture electron microscopy, changes in the lamellar organization in stratum corneum of both patient groups could be observed.Keywords: ceramides, cryoelectron microscopy, diseased human skin, electron diffraction, freeze fracture
UV irradiation induces a variety of cutaneous responses, including disruption of epidermal permeability barrier function, the basis for which is not known. Herein, we investigated the separate roles of hyperproliferation and inflammation in the pathogenesis of UVB-induced barrier disruption. Adult hairless mice were exposed to increasing doses of UVB (1.5–7.5 MED), and transepidermal water loss (TEWL) was monitored daily for up to 7 d. The extent of TEWL increase was dependent on the UVB dose, but with all doses, the increase began after 48 h and peaked at 96 h, decreasing by 120 h. Epidermal [³H]thymidine incorporation increased at 24 h and peaked at 48 h (570%), preceding the maximal increase in TEWL. Cyclosporin A, methotrexate, 5-fluorouracil, or arabinosylcytosine significantly diminished the UVB-induced TEWL increase. Athymic nude mice also displayed a markedly diminished response to UVB, and DNA synthesis did not increased at 48 h. Transplantation of athymic mice with T-cell- enriched mixed immune cells significantly restored sensitivity to both the UVB-induced hyperproliferation and the barrier defect. Finally, although UVB exposure increased PGE2 levels in whole skin samples (2- to 3-fold within 1–3 h; p < 0.005), this increase was completely blocked by topical indomethacin, and neither topical indomethacin nor topical glucocorticoids blocked development of the barrier abnormality. These results show that (i) UVB produces delayed alteration in barrier function and (ii) both an epidermal proliferative response and thymocyte-mediated events (but not PGE2 production and nonspecific inflammation) appear to contribute to UVB-induced abrogation of the permeability barrier.Keywords: epidermal permeability barrier, DNA synthesis, T cells, athymic
132 psoriasis patients who had been completely cleared with dithranol were assessed and then followed up over a 3-year period. The incubation time from specific incidents of stress to the development of psoriasis was between 2 days and 1 month. Specific stress within a month before the first attack was recalled by 51 patients (39%); chance association was excluded by studying a control group where there was significantly less specific stress. The prognosis for the psoriasis patients who recollected specific stress a month prior to the onset was significantly better than for the rest of the patients where none had been recalled.
Covalently bound lipids have been identified and compared in keratinizing porcine epithelia including epidermis and oral epithelium from palate and gingiva. Stratum corneum was isolated by tryptic digestion, and after extensive extraction of lipids using a series of chloroform-methanol mixtures, the residual tissue was subjected to alkaline hydrolysis to release covalently bound lipids. The lipids so released were analyzed by quantitative thin-layer chromatography. Stratum corncum from each of the three anatomical sites contained ω-hydroxyceramides, ω-hydroxyacids and fatty acids. In epidermal stratum corneum the total covalently bound lipids represented 2.4% of the dry weight of the tissue, but in the oral epithelia this figure was consistently lower: 0.24% in palatal stratum corneum and 0.20% in gingival stratum corneum. Transmission electron microscopy before and after lipid extraction confirms the presence of a lipid envelope in epidermal stratum corneum and demonstrates the absence of this structure in oral stratum corneum.
Exposure of human keratinocytes to UVA radiation induced an increase in ceramide (CER) intracellular content, with a dose-dependent effect within the range of 4–9 J/cm2. The production of CER reached a maximum 2 h after UVA irradiation. The increase of CER was proportional to the intracellular content of reactive oxygen species, was prevented by the antioxidant vitamin E, and enhanced by the prooxidant buthionine-sulfoximine, suggesting the involvement of an oxidative stress. UVA decreased both neutral and acid sphingomyelinase activities measured in vitro. A direct cleavage of sphingomyelin to CER by UVA, recently described, was not observed under our experimental conditions. We also show that, downstream of CER, UVA activated the Ser/Thr kinases ERK, JNK, and p38. Since ceramide has been shown to play a role in stress kinase activation, our results provide a possible mechanism for UVA-induced activation of stress kinases via ceramide formation. However, the actual mechanisms whereby CER is produced in cultured cells under UVA exposure remain to be specified.
A phenomenon of increased permeation as a function of time in the percutaneous absorption of urea and water led to this in vitro study with skin of hairless mice in a closed diffusion cell system. The Latin square method was used to assess the effect of four variables, namely temperature, time of hydration, concentration of urea and stirring of the donor-and receiver-cell phases on the physical structure of the skin and the permeability coefficients of urea and water. The scanning electron microscope and the transmission electron microscope were used to assess the physical changes in skin structure.
Temperature and time of hydration, but not the concentration of urea or the stirring of the donor- and receiver-cell phases, affected the permeability of the skin to urea and water. The scanning electron microscope and transmission electron microscope micrographs confirmed these results.
L'observation d'une pénétration accélérée dans le temps dans le phénomène d'absorption percutanée de l'urée et de l'eau a conduit à cette étude in vitroà l'aide de peau de souris nue dans un système de diffusion cellulaire fermé. L'analyse de variances a été utilisée pour évaluer l'effet de quatre variables, notamment la température, le temps d'hydratation, la concentration de l'urée et le mouvement des phases cellulaires du donneur et du receveur sur la structure physique de la peau et les coefficients de perméabilité de l'urée et de l'eau. Le microscope électronique d'exploration et le microscope électronique de transmission ont été utilisés pour évaluer les changements physiques intervenant dans la structure de la peau.
Seuls la température et le temps d'hydratation ont affecté la perméabilité de la peau à l'urée et à l'eau alors que la concentration en urée et le mouvement des phases cellulaires du donneur et du receveur n'ont eu aucun effet.
A retrospective assessment of insight was undertaken in 61 patients whose psoriasis was considered to have been precipitated by stress. A significantly better prognosis was observed in those patients assessed as having had insight.
Stratum corneum sphingolipids are of particular importance in maintaining the water permeability barrier of mammalian epidermis. Free amino acids also play an important role in water retention in the stratum corneum. To clarify the way in which these substances affect scaly skin, stratum corneum sphingolipids and free amino acids collected from artificially-induced scaly skin were analysed. Scaly skin was induced by tape stripping. The total amount of sphingolipids was quantified by gas chromatography and five of sphingolipid fractions were isolated and quantified by thin-layer chromatography. Free amino acids were analysed using a high-speed amino analyser. The total amount of sphingolipid in scaly skin did not differ statistically from that in control skin. However, a significant change in the distribution of the five sphingolipid species was observed in scaly skin and the total amount of amino acids was decreased in scaly skin. These results suggest that the distribution of these five types of sphingolipid and the total amount of amino acids are responsible for scaly skin.
Reactive changes produced in human epidermis by occlusion with water for 24 or 48 h were studied. A focally widened intercellular space was common. Several, often pronounced, reactive events were observed in the Langerhans cell system, whereas the keratinocytes and the melanocytes seemed unaffected. The reactive events showed a scattered distribution and were revealed only by electron microscopic analysis of extensive section series. It seems that, among epidermal cells, the Langerhans cells are the most susceptible and most easily alerted by an exogenous challenge.
The local sweating response to thermal stress (mean ambient temperature 33 degrees C) was assessed under resting conditions on the non-eczematous back skin of 26 young men with atopic dermatitis (AD) and in 22 non-atopic controls with other dermatoses. The baseline (transepidermal) water loss was separately determined at room temperature (mean 23.6 degrees C) to calculate the pure sweat loss. A gravimetric collecting method was used for the measurements at 40, 60 and 80 min. In the heated room the sweat loss in AD patients was significantly lower at all time intervals. The cumulative sweat loss was 50-60% lower in AD patients than in the controls (P less than 0.02). Subjects with dry AD skin had a lower sweat loss than subjects with normal-looking skin. Compared with controls the sweat loss in AD patients was lowest at 40 min, suggesting a retarded sweating response. Half of the patients with AD and half of the controls had active participation in sports, and showed a greater sweat loss compared to the non-sporting subjects in the same group.
This chapter discusses the biochemistry and function of stratum corneum lipids. The study of lipids as a class of chemical constituents of the stratum corneum offers a unique opportunity to investigate the functional specialization of this tissue. The daily rate of epidermal lipid synthesis in man is equal to the lipid content times the daily loss of stratum corneum. Total epidermal lipid constitutes approximately 10–14% of the dry weight of mammalian epidermis. However, by themselves, isolated intercellular lipids possess no water-holding capacity. The ability of the intercellular lipids to form lamellar bilayers, in the absence of phospholipids, is dependent upon the amphipathic properties of ceramides, free fatty acids, cholesterol, and perhaps lesser constituents such as cholesterol sulfate and proteolipids. The lamellar bilayers are stabilized in an aqueous environment by van der Waals interactions and hydrogen bonds. Moreover, recently, it has been suggested that a major component of the stratum corneum is a ceramide, consisting of 30 to 34-carbon chain length, N-acyl, ω-hydroxyacids covalently bound to the cornified envelope. This leaflet may serve as a scaffold for the intercellular bilayers, thereby contributing to both the barrier and the cohesive properties of the stratum corneum.
As reviewed in this article, the stratum corneum must now be accorded the respect due to a structurally heterogeneous tissue possessing a selected array of enzymatic activity. The sequestration of lipids to intercellular domains and their organization into a unique multilamellar system have broad implications for permeability barrier function, water retention, desquamation, and percutaneous drug delivery. Yet, the functions and organization of specific lipid species in this membrane system are still unknown. Certain novel insights have resulted from comparative studies in avians and marine mammals. Further elucidation of the molecular architecture and interactions of lipid and nonlipid components of the stratum corneum intercellular domains will be a prerequisite for a comprehensive understanding of stratum corneum function.
Stratum corneum lipids are an important determinant for both water-retention function and permeability-barrier function in the stratum corneum. However, their major constituent, ceramides, have not been analyzed in detail in skin diseases such as atopic dermatitis that show defective water-retention and permeability-barrier function. In an attempt to assess the quantity of ceramides per unit mass of the stratum corneum in atopic dermatitis, stratum corneum sheet was removed from the forearm skin by stripping with cyanoacrylate resin and placed in hexane/ethanol extraction to yield stratum corneum lipids. The stratum corneum was dispersed by solubilization of cyanoacrylate resin with dimethylformamide, and after membrane filtration, the weight of the stratum corneum mass was measured. The ceramides were quantified by thin-layer chromatography and evaluated as microgram/mg stratum corneum. In the forearm skin of healthy individuals (n = 65), the total ceramide content significantly declined with increasing age. In atopic dermatitis (n = 32-35), there was a marked reduction in the amount of ceramides in the lesional forearm skin compared with those of healthy individuals of the same age. Interestingly, the non-lesional skin also exhibited a similar and significant decrease of ceramides. Among six ceramide fractions, ceramide 1 was most significantly reduced in both lesional and non-lesional skin. These findings suggest that an insufficiency of ceramides in the stratum corneum is an etiologic factor in atopic dry skin.
In order to examine the time course of lipid metabolism in the epidermis, pairs of sites on each of two pigs were given seven intradermal injections of 10 μCi of [1 -¹⁴C] -acetate at 1, 3, 6, 12, 24, 36, and 48 h before the animals were killed. A second time course was obtained with two other pigs at 1 h and 3.5, 7, 10.5, 14, 17.5, and 21 d after labeling. The epidermis over each injection site was recovered, and the lipids were extracted and analyzed by thin-layer chromatography and by autoradiography.
Virtually all of the radioactivity in aliphatic chains was found first in phospholipids and was then transferred to glucosylceramides, and then into ceramides, between 1 and 7 d after labeling. Labeling of free fatty acids showed a similar time course. Most of the radioactivity acquired by acylglucosylceramide was transferred to hydroxyceramide, bound to the corneocyte envelope, between 2 and 10 d after labeling. The hydroxyacid and sphingosine moieties of the hydroxy- ceramide had equal specific activity. The results indicate that the aliphatic lipids of the stratum corneum are synthesized de novo in the epidermis via phospholipids. Cholesterol, however, was synthesized directly from acetate within hours of labeling, and its esters acquired label 3 to 7 d later.
UVA, in high-dose single exposures, can, like UVB, be deleterious to skin. Dermal damage resulting from chronic exposure to UVA has not been studied. To investigate the long-term effects, we irradiated albino hairless mice for 30-34 weeks with UVA radiation, alone, from two sources with differing spectral qualities, and in combination with UVB as solar-simulating radiation. The results were compared to UVB alone.
Like UVB, the UVA waveband, especially that with a spectral distribution similar to solar UVA, caused elastic fiber damage, increased glycosaminoglycan levels, and produced hypertrophy of deep dermal tissues. There were, however, striking differences between UVB- and UVA-irradiated skin. A combination of UVA and UVB summated the effects of both wavebands. Substantial protection against these effects was afforded by a broad-spectrum sunscreen.
The permeation of compounds through skin damaged by different methods was compared because agents that are absorbed through skin are sometimes applied to a damaged barrier. The removal of the stratum corneum by stripping the skin with cellophane tape was the most effective method for enhancing absorption. A minimal increase in water permeation was obtained when one abrasion line was made with a hypodermic needle, but the absorption increased substantially when three to six lines were made across the site of application. Similar values were obtained with in vivo and in vitro techniques for penetration of cortisone and nicotinic acid through normal and abraded rat skin. Sever damage by UV irradiation to rats in vivo resulted in nicotinic acid absorption similar to that obtained in vitro through abraded or tape-stripped skin. Damage from mild irradiation could not be accurately duplicated by in vitro methods. The magnitude of the increase in absorption of seven chemicals through abraded human and rat skin was related to the extent to which the molecules were absorbed by the skin. The greatest increases in penetration were obtained with the compounds that were most poorly absorbed.
The carcinogenic effect of UVA radiation (from Philips black light tubes filtered through a 2 mm-thick glass plate to eliminate the radiation below 320 nm) was studied in 7 groups of 25 lightly pigmented hairless mice. Irradiation with a moderate daily dose of combined UVB and UVA for 3 months induced a tumor incidence of 0.22 after 58 weeks. When the combined UVB and UVA irradiation was followed by filtered UVA for 2, 4, or 6 months, the tumor incidence was marginally significantly increased to 0.42, 0.48, and 0.50 (p less than 0.05), respectively. However, irradiation with the moderate dose of combined UVB and UVA induced a slight but not significantly lower tumor incidence as compared to UVB alone (0.22 vs 0.30, p greater than 0.1). UVA alone induced no tumors. It thus appears that in hairless mice initially exposed to a combination of UVB and UVA, subsequent continued irradiation with UVA increases tumor incidence. While only marginally statistically significant, tumor incidence in these animals seems to increase with duration and hence total UVA exposure. Furthermore, it is suggested that the photoaugmentative carcinogenic effect of UVA irradiation from unfiltered UVA bulbs can be reduced by attenuating the shorter wavelengths of the radiation.
Hydrolysis of glucosylceramides (GlcCer) by beta-glucocerebrosidase generates ceramides, critical components of the epidermal permeability barrier. Ceramides also are involved in the regulation of cellular proliferation and differentiation in a variety of cell types. Whereas most studies have focused on ceramides and their sphingoid base metabolites as growth inhibitors, GlcCer apparently acts oppositely (i.e., as a mitogen). To determine whether enhancement of GlcCer content stimulates epidermal mitogenesis, we examined the response of hairless mouse epidermis to alterations in endogenous and/or exogenous GlcCer. Topical applications of conduritol B epoxide, a specific irreversible inhibitor of beta-glucocerebrosidase, increased epidermal GlcCer levels twofold, an alteration localized largely to the basal, proliferative cell layer (fourfold increase); and stimulated epidermal proliferation (2.3-fold elevation in [3H]thymidine incorporation; P < or = 0.001), localized autoradiographically again to the basal layer, and resulting in epidermal hyperplasia. Intracutaneous administration of GlcCer (2.0 mg) also stimulated epidermal DNA synthesis, while simultaneous treatment with conduritol B epoxide plus GlcCer resulted in an additive increase in DNA synthesis. These increases in epidermal proliferation could not be attributed either to altered epidermal permeability barrier function, or to nonspecific irritant effects, as determined by four separate criteria. These results strongly suggest that GlcCer directly stimulates epidermal mitogenesis.
To elucidate the mechanisms that are involved in the decrease of ceramide levels in atopic dry skin and in aged skin, we examined both the activities of beta-glucocerebrosidase, which is a major enzyme in ceramide production, and of ceramidase, which is an essential enzyme in ceramide degradation, in the stratum corneum of atopic dry skin and aged skin. The specimens of the stratum corneum of forearm skin were obtained by tape-stripping from 61 healthy volunteers and 23 patients with atopic uninvolved skin. The beta-glucocerebrosidase activity in the stratum corneum extracts was estimated using fluorescent 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate. Ceramidase activity was determined using 14C-palmitoylsphingosine as the substrate. Among the atopic skin samples, neither beta-glucocerebrosidase nor ceramidase activities were different from those of age-matched healthy controls. Nor was the beta-glucocerebrosidase activity deficient in the aged skin samples as compared to that seen in samples from the young, healthy group. In contrast, there was an age-related upregulation in ceramidase activity. The results indicate that the decrease of ceramides in atopic dry skin may not be accompanied by reduced synthesis or by enhanced degradation, each of which is primarily attributable to the above two enzymes, respectively. The pathogenesis of aged dry skin can be explained, at least partially, in terms of elevated ceramidase activity, which results in a disturbance of the lamellar structure of the stratum corneum lipids.
It is well established that exposure to solar UVB (290-320 nm) gives rise to mutations in oncogenes and tumor suppressor genes that initiate the molecular cascade toward skin cancer. Although UVA (320-400 nm) has also been implicated in multistage photocarcinogenesis, its potential contribution to sunlight mutagenesis remains poorly characterized. We have determined the DNA sequence specificity of mutations induced by UVB (lambda > 290 nm), and by UVA (lambda > 350 nm), at the adenine phosphoribosyltransferase locus of Chinese hamster ovary cells. This has been compared to results previously obtained for stimulated sunlight (lambda > or = 310 nm) and 254-nm UVC in the same gene. We demonstrate that T-->G transversions, a generally rare class of mutation, are induced at high frequency (up to 50%) in UVA-exposed cells. Furthermore, this event comprises a substantial proportion of the simulated sunlight-induced mutant collection (25%) but is significantly less frequent (P < 0.05) in cells irradiated with either UVB (9%) or UVC (5%). We conclude that the mutagenic specificity of broad-spectrum solar light in rodent cells is not determined entirely by the UVB component and that UVA also plays an important role.
This paper investigates the ceramide composition of the psoriatic scale compared with that of normal human SC. A method was optimalized, based on TLC separation followed by densitometry, allowing the provision of good resolution and quantification of ceramide fractions from both normal and pathological specimens. Seven ceramide fractions were isolated and submitted to compositional analysis. The obtained results suggested a revisitation of previous ceramide designation. Therefore a simple classification is suggested, based on grouping ceramides carrying structural similarities under common codes. According to these rules, ceramides were grouped into five classes designated as: (1) Cer[EOS], which contains ester-linked fatty acids, omega-OH fatty acids and sphingosines; (2) Cer[NS], which contains non-OH fatty acids and sphingosines; (3) Cer[NP], which contains non-OH fatty acids and phytosphingosines; (4) Cer[AS], which contains alpha-OH fatty acids and sphingosines; (5) Cer[AP], which contains alpha-OH fatty acids and phytosphingosines. Analysis of ceramides from the psoriatic scale, compared to those from normal human SC, resulted in an impairment of the Cer[EOS] content as well as of the ceramides containing phytosphingosine, with concurrent increase in ceramides containing sphingosine, being the total amount maintained identical. Since one of the suggested pathways for phytosphingosine biosynthesis involves the water addition to the corresponding sphingosine double bond, we can speculate that the observed alteration is due to a deranged water bioavailability, associated with psoriasis.
We examined the possibility that the cutaneous permeability barrier regulates epidermal DNA synthesis in two acute and two chronic models of barrier perturbation. In animals treated topically with acetone, DNA synthesis is increased 102%, in tape-stripped animals 127%, in essential fatty acid deficient animals 50%, and in animals chronically treated with topical lovastatin 64%. This linkage between disturbances in barrier function and increased DNA synthesis is further supported by specific and correlative observations: (a) in these disparate models, artificial replacement of the barrier with a water-impermeable membrane inhibits the expected increase in DNA synthesis; (b) the extent of the burst in DNA synthesis is proportional to the degree of barrier abrogation; (c) the inhibition of DNA synthesis by membranes is directly related to the degree of permeability of these occlusive membranes, i.e., the more impermeable the greater the degree of inhibition; (d) topical treatment with lipids that restore barrier function corrects the increase in DNA synthesis; and (e) barrier abrogation with acetone produces an increase in epidermal DNA synthesis without altering bulk protein synthetic rates in contrast to events known to follow injury or cell replacement. Autoradiographic studies show that the increase in DNA synthesis after acetone treatment is limited to the epidermal basal layer. This constellation of findings strongly suggests that cutaneous barrier function is one factor that regulates epidermal DNA synthesis.
Work in the past 8 years, particularly in the past 1-2 years, has greatly expanded our understanding of the mechanisms by which ultraviolet irradiation stimulates melanogenesis in the skin. A direct effect of UV photons on DNA results in up-regulation of the gene for tyrosinase, the rate-limiting enzyme in melanin synthesis, as well as an increase in cell surface expression of receptors for at least one of the several known keratinocyte-derived melanogenic factors, MSH. Direct effects of UV on melanocyte membranes, releasing DAG and arachidonic acid, may also play a role in the tanning response. Diacylglycerol may activate PKC-beta, which in turn phosphorylates and activates tyrosinase protein; the pathways by which products of other inflammatory mediator cascades may act on melanogenesis are unknown. The tanning response also relies heavily on UV-stimulated increased production and release of numerous keratinocyte-derived factors including bFGF, NGF, endothelin-1 and the POMC-derived peptides MSH, ACTH, beta-LPH and beta-endorphin. These factors variably induce melanocyte mitosis, increase melanogenesis, enhance dendricity and prevent apoptotic cell death following the UV injury. Thus, events within the epidermal melanin unit conspire to maintain or increase melanocyte number, increase melanin pigment throughout the epidermis. Overall, ultraviolet-induced melanogenesis may be one part of a eukaryotic SOS response to damaging ultraviolet irradiation that has evolved over time to provide a protective tan in skin at risk of further injury from sun exposure. These recent insights into the mechanisms underlying ultraviolet-induced melanogenesis offer the opportunity for novel therapeutic approaches to minimizing acute and chronic photodamage in human skin.
Three stratum corneum lipids, ceramides, cholesterol (CHOL), and free fatty acids (FA), are required for permeability barrier homeostasis. Recent studies have shown that application of one or two of these lipids to perturbed skin delays barrier recovery; only equimolar mixtures allow normal recovery. We asked here whether any physiological lipid mixtures improve barrier repair, as assessed by transepidermal water loss. Whereas an equimolar ratio of ceramides, CHOL, and FA (either the essential fatty acid, linoleic acid, or the nonessential FAs, palmitic or stearic acids) allows normal repair, further acceleration of barrier repair occurs as the ratio of any of these ingredients is increased up to 3-fold. Similar preliminary results were obtained in damaged human skin. Likewise, while acylceramides alone delay barrier recovery, acylceramides: CHOL mixtures within a specific range of molar rations dramatically improve barrier repair. Furthermore, glycosyl ceramides, sphingomyelin, and triglycerides substitute effectively for ceramides and FA, respectively, but neither phospholipids nor cholesterol esters substitute for FA and CHOL, respectively. These studies show the specific requirements of selected stratum corneum lipid mixtures for optimized barrier repair in murine skin, with further validation in human skin. Utilization of physiologic lipids according to these parameters could lead to new forms of topical therapy for dermatoses (e.g., psoriasis, atopic dermatitis, and irritant dermatitis) triggered by abnormal barrier function.
Sodium lauryl sulfate (SLS), a surfactant frequently used in the induction of experimental irritant contact dermatitis in animals and in humans, characteristically induces a dose-related increase in TEWL (transepidermal water loss). Ceramides are considered to be important in the regulation of the skin barrier. We therefore examined the relationship between initial ceramide content of stratum corneum and induced changes in skin color (erythema) and barrier function, after SLS application under occlusion (1% and 3% in water) to the forearm of 14 volunteers. Stratum corneum sheets were removed, stratum corneum lipids extracted, and ceramide composition determined from chromatograms (TLC) using densitometry. After determining baseline skin color and TEWL at each area, 2 samples of stratum corneum were obtained from each volunteer. Clinical and instrumental controls of the SLS-induced irritation were performed at 24, 48, 72 and 96 h. Erythema was evaluated by colorimetry: barrier impairment by changes in TEWL. We found inverse correlations between baseline ceramide 61 (weight) and the 24 h erythema score for SLS 3%, between ceramide 1 and 24 h TEWL, and between ceramide 611 and 72 h TEWL for SLS 3%. Our findings suggest that low levels of these ceramides may determine a proclivity to SLS-induced irritant contact dermatitis.
Stratum corneum lipids play a predominant role in maintaining the water barrier of the skin. In order to understand the biological variation in the levels and composition of ceramides, ceramide 1 subtypes, cholesterol and fatty acids, stratum corneum lipids collected from tape strippings from three body sites (face, hand, leg) of female Caucasians of different age groups were analysed. In addition, we studied the influence of seasonal variation on the lipid composition of stratum corneum from the same body sites. The main lipid species were quantified using high-performance thin-layer chromatography and individual fatty acids using gas chromatography. Our findings demonstrated significantly decreased levels of all major lipid species, in particular ceramides, with increasing age. Similarly, the stratum corneum lipid levels of all the body sites examined were dramatically depleted in winter compared with spring and summer. The relative levels of ceramide 1 linoleate were also depleted in winter and in aged skin whereas ceramide 1 oleate levels increased. The other fatty acid levels remained fairly constant with both season and age, apart from lignoceric and heptadecanoic acid which showed a decrease in winter compared with summer. The decrease in the mass levels of intercellular lipids and the altered ratios of fatty acids esterified to ceramide 1, are likely to contribute to the increased susceptibility of aged skin to perturbation of barrier function and xerosis, particularly during the winter months.
Age of onset of skin cancers in normal and xeroderma pigmentosum (XP) skin cancer patients. The cumulative percentage of patients with BCCs or SCCs of the skin is plotted versus the age at diagnosis. The curve for the normal population is based on 29,757 skin cancers surveyed by the National Cancer Institute (1). The curve for the xeroderma pigmentosum patients is based on 63 skin cancers reported to the Xeroderma Pigmentosum Registry (unpublished data).
Ultraviolet (UV) irradiation of the skin induces a variety of responses in the epidermis, including sunburn cell formation, epidermal hyperplasia, and a dose-dependent permeability barrier abnormality, an effect that appears to be dependent upon both UVB-induced hyperplasia and T-cell activation. Since intrinsically aged epidermis displays decreased epidermal turnover, diminished inflammatory response to various stimuli, including UVR, and impaired immune function, we investigated the effects of UVB on both epidermal barrier function and proliferation in hairless mice of increasing chronologic age (27, 61, and 90 wk). After a single UVB exposure (0.15 J/cm2 7.5 MED), a barrier abnormality developed (i.e., increased transepidermal water loss; TEWL), after a delay of > or = 48 h, regardless of age. In young mice (27 wk old), TEWL levels peaked at 72-96 h (9.9-fold over untreated controls), whereas increased epidermal [3H]thymidine incorporation preceded the peak TEWL increase (i.e., approximately 570% increase over controls at 48 h). In contrast, the UVB-induced increased in both TEWL and DNA synthesis were significantly diminished, with decreased epidermal hyperplasia evident, in intrinsically aged versus young mouse epidermis. Baseline epidermal thickness decreased with animal age (i.e., 16.8 +/- 3.1 vs. 27.9 +/- 0.7 microm for 90- vs. 27-wk-old animals, respectively; p < 0.02), suggesting that the diminished barrier response with aging reflects an attenuation of events subsequent to initial UVB exposure, rather than an increase in the UV dose delivered. These results demonstrate that (i) murine epidermis becomes less sensitive to UVB-induced barrier alterations with age and (ii) decreased DNA synthesis after UVB correlates with the age-related decrease in barrier dysfunction.
The sphingomyelin pathway is a ubiquitous, evolutionarily conserved signaling system initiated by hydrolysis of the plasma membrane phospholipid sphingomyelin to generate the second messenger ceramide. Sphingomyelin degradation is catalyzed by acid and neutral sphingomyelinase (SMase) isoforms. Most, if not all mammalian cells, appear capable of signaling though the sphingomyelin pathway. Diverse receptor types and environmental stresses utilize the sphingomyelin pathway as a downstream effector system. In some cellular systems, ceramide initiates differentiation or cell proliferation, while in other systems, ceramide signals apoptosis. Recent investigations link the activation of neutral SMase to the extracellular signal regulated kinase (ERK) cascade and pro-inflammatory responses, and acid SMase to the stress-activated protein kinase/c-jun kinase (SAPK/JNK) cascade and apoptotic responses. Environmental stresses act directly on membrane to activate acid pH-dependent sphingomyelinase (ASMase), whereas cytokine receptors signal ASMase activation through motifs termed death domains. The present review focuses on mechanisms of activation of ASMase and on ceramide signaling of the apoptotic response.
The sequence of events affecting skin morphogenesis occurring after a single exposure to UVB was investigated on a model of human skin reconstructed in vitro. The biologically efficient dose (BED) able to induce the early UVB-DNA damages such as pyrimidine dimers, sunburn cells, and apoptotic keratinocytes was determined as 50 mJ/cm2. The subsequent changes induced during a period of 14 days following irradiation were analyzed. Up to Day 3, an epidermal disorganization led to a parakeratotic epidermis characterized by nucleated horny layers, as well as the down regulation of major markers of keratinocyte differentiation such as keratin 10, loricrin, filaggrin, and the keratinocyte transglutaminase (type I). On the contrary, the expression of involucrin and spr1 seemed to be unaffected, indicating distinct responses to UVB of proteins involved in keratinocyte differentiation. A progressive regeneration of normal epidermal morphogenesis begins from Day 4 leading to the normalization of keratinocyte differentiation at Day 10 to 14. In parallel, epidermal proliferation was increased. Taken together, these findings show that in skin reconstructed in vitro, UVB exposure leads to major epidermal developmental changes characterized by (i) an early apoptotic process, (ii) a subsequent down-regulation of specific keratinocyte differentiation markers, and (iii) the recovery of both the early and delayed effects resulting in normal epidermal morphogenesis.
The stratum corneum of vertebrates is a major structural compartment that provides mechanical protection and prevents skin desiccation. The water barrier function of the stratum corneum was first reported in 1944, and this was shown later to be associated with multilayered lipid lamellae localized in the extracellular spaces. The major lipid components isolated from the cornified epidermal layers are ceramides, which belong to the class of sphingolipids, cholesterol and free fatty acids; their biosynthesis is in tight relationship with the cutaneous barrier function. In studies in which the barrier is artificially disturbed, lipid biosynthesis is found to be directly regulated by barrier permeability. As mentioned above, the ceramides involved in this process are located in the extracellular spaces of the upper epidermal layers, whereas sphingomyelin, the most common sphingolipid, is an integral part of the bilayer plasma membrane of the keratinocytes. During the last few years, however, increasing evidence has shown that sphingolipids may also take part in cell signalling, and the term 'sphingomyelin cycle' has been coined to describe this novel path-way of signal transduction. Intracellular messengers of the sphingomyelin cycle are ceramides as the products of an agonist-stimulated sphingomyelin hydrolysis. Increased levels of intracellular ceramides induce cell differentiation and/or apoptosis and reduce cell proliferation. In contrast to the extracellular barrier-forming ceramides which are complex partly O-acylated species containing long-chain fatty acids, intracellular signal-transducing ceramides are not O-acylated and have acyl chain lengths of 16 and 18 carbon atoms. We present here a review of our present knowledge on the sphingomyelin cycle as a possible signal transduction pathway in the human epidermis. We discuss the common origin of extracellular ceramides constituting the lipid barrier and of intracellular ceramides generated by agonist-stimulated sphingomyelin hydrolysis and serving as second messengers. A summary of alterations in sphingolipid metabolism and lipid composition of the epidermis in diseased skin is also given and the possible use of different sphingolipids for therapy is envisaged.
The extracellular lipids of the stratum corneum, which are comprised mainly of cholesterol, fatty acids, and ceramides, are essential for epidermal permeability barrier function. Moreover, disruption of the permeability barrier results in an increased cholesterol, fatty acid, and ceramide synthesis in the underlying epidermis. This increase in lipid synthesis has been shown previously to be due to increased activities of HMG-CoA reductase, acetyl-CoA carboxylase, fatty acid synthase and serine palmitoyl transferase, key enzymes of cholesterol, fatty acid, and ceramide synthesis, respectively. In the present study, we determined whether the mRNA levels for the key enzymes required for synthesis of these three classes of lipids increase coordinately during barrier recovery. By northern blotting, the steady-state mRNA levels for HMG-CoA reductase, HMG-CoA synthase, farnesyl pyrophosphate synthase, and squalene synthase, key enzymes for cholesterol synthesis, all increased significantly after barrier disruption by either acetone or tape stripping. Additionally, the steady-state mRNA levels of acetyl-CoA carboxylase and fatty acid synthase, required for fatty acid synthesis, as well as serine palmitoyl transferase, the rate-limiting enzyme of de novo ceramide synthesis, also increased. Furthermore, artificial restoration of the permeability barrier by occlusion after barrier disruption prevented the increase in mRNA levels for all of these enzymes, except farnesyl pyrophosphate synthase, indicating a specific link of the increase in mRNA levels to barrier requirements. The parallel increase in epidermal mRNA levels for the enzymes required for cholesterol, fatty acid, and ceramide synthesis may be due to one or more transcription factors that regulate lipid requirements for permeability barrier function in keratinocytes.
Ultraviolet light (UVR) induces a myriad of cutaneous changes, including delayed disruption of the permeability barrier with higher doses. To investigate the basis for the UVB-induced barrier alteration, we assessed the epidermal lamellar body secretory system at various time points before and after barrier disruption with a single high dose of UVB (7.5 MED) to murine epidermis. Morphological data were correlated with changes in epidermal proliferation and lipid synthesis, indicative of lamellar body generation. Twenty-four hours following UVB, the stratum corneum (SC) is normal, but a layer of abnormal, vacuolated, and lamellar body (LB)-deficient cells is present, immediately beneath the stratum granulosum (SG)/SC interface. Immediately subjacent to this band of damaged cells, normal keratinocytes that contain intact LBs are present. By 72 h, concomitant with the appearance of a barrier abnormality, extensively damaged cells persist at the SC/SG interface, and abnormal lamellar membrane structures appear in the lower SC. Upper stratum spinosum (SS) and lower SG cells appear normal, with increased numbers of LBs. A barrier abnormality is still present at 96 h, in association with membrane abnormalities in the lower SC interstices, but up to four normal appearing, subjacent SG cell layers are present. By 120 h, accelerated LB formation and precocious LB extrusion occur throughout the thickened SG; normal lamellar membranes are present in the lower SC; and barrier recovery is almost complete. Whereas, epidermal synthesis of the major barrier lipid species (i.e., cholesterol, fatty acids, and ceramides, including acylceramides) is reduced or unchanged at 24 and 48 h, it increases significantly 72 h after exposure to UVB. Therefore, the delayed disruption of the permeability barrier following acute UVB exposure results from the arrival of a band of lamellar body-incompetent (i.e., damaged) cells at the SG/SC interface. The subsequent, rapid recovery of the barrier, in turn, results from compensatory hyperplasia of subjacent, undamaged SS/SG cells, generating increased numbers and contents of LB. These results underscore the critical role of the stratum compactum in mediating barrier function, and suggest that beneficial therapeutic effects of UV exposure may be due to enhanced lipid production and barrier regeneration.
To examine the effect of stress on skin homeostasis, cutaneous barrier recovery was measured in rate exposed to immobilization stress after tape stripping or sodium dodecyl sulphate treatment. The barrier function was evaluated by measuring transepidermal water loss. Barrier recovery was delayed in rats exposed to stress in comparison with untreated controls. This tendency was observed in both male and female animals. The delay in barrier recovery was blocked by application of the sedative drugs diazepam and chlorpromazine. The barrier recovery rate in mice which were kept at a high population density (10 animals per cage) for 2 weeks was slower than that in mice kept at lower population densities (five animals or one animal per cage). These animal models could be useful for objectively quantifying the influence of stress on the cutaneous function.