Water extract of gromwell (Lithospermum erythrorhizon) enhances migration of human keratinocytes and dermal fibroblasts with increased lipid synthesis in an in vitro wound scratch model.

BACKGROUND: Although organic extracts of gromwell (Lithospermum erythrorhizon) have been shown to promote wound healing, the wound healing effects of water extracts of gromwell (WG) that are commonly used in traditional remedies have not been elucidated. OBJECTIVE: We investigated whether WG promotes the migration and/or proliferation of cultured human keratinocytes (CHK) or dermal fibroblasts in parallel with increases in lipid synthesis during in vitro wound healing. METHODS: CHK or fibroblasts were treated with 1-1,000 µg/ml WG for up to 48 h following scratch wound formation. Cell migration was assessed by measuring coverage (in percent) from the wound margin, while cell proliferation and lipid synthesis were determined by [(3)H]thymidine incorporation into DNA fractions, and [(3)H]palmitate or [(3)H]serine incorporation into lipid fractions, respectively. RESULTS: Low-dose WG (1 µg/ml) enhanced the wound coverage for both CHK and fibroblasts at 24 h, while cell proliferation was not altered in either cell types. Synthesis of both total lipids and individual lipid classes, including phospholipids, sphingolipids and neutral lipids, were found to be increased at 24 h in CHK treated with 1 µg/ml WG; in similarly treated fibroblasts, only the syntheses of sphingolipids (such as ceramides and glucosylceramides), but not other lipid species, were significantly increased. In contrast, a higher dose of WG (10-1,000 µg/ml) did not enhance wound coverage, and 100 µg/ml WG neither altered cell proliferation nor lipid synthesis in both CHK and fibroblasts. CONCLUSION: Low-dose WG (1 µg/ml) enhances the migration of both CHK and fibroblasts with increased lipid synthesis in an in vitro wound scratch model.

Kinetic characteristics of acidic and alkaline ceramidase in human epidermis.

It has recently become evident that at least five ceramidase (CDase) isoforms are present in human epidermis, and that specifically acidic CDase (aCDase) and alkaline CDase (alkCDase) activities increase during keratinocyte differentiation, and thus might play a pivotal role(s) in permeability barrier function. Prior to investigating their possible roles in the epidermal barrier function, it is necessary to characterize basic kinetic parameters for these enzymes, as well as to determine the effects of the established CDase inhibitors and their activities. In this study, assays for both aCDase and alkCDase activities in fully differentiated human epidermis were optimized using a radiolabeled substrate. These studies revealed that aCDase activity is substantially higher than alkCDase activity, and that both isoenzymes are inhibited by a CDase inhibitor N-oleylethanolamine. These findings were also confirmed using an in situ enzyme assay.

Vitamin C stimulates sphingolipid production and markers of barrier formation in submerged human keratinocyte cultures.

Human keratinocytes differentiate in vitro in response to a variety of stimuli, but neither the levels nor the spectrum of ceramides approach those seen in vivo. Ceramide production increases when human keratinocytes are grown at an air-liquid interface, and alterations in ceramide content occur when vitamin C is added to air-exposed, organotypic culture systems (Ponec et al. J Invest Dermatol 109:348, 1997). Here, we assessed whether vitamin C stimulates sphingolipid production in human keratinocytes independent of differentiation and air exposure. When submerged, human keratinocytes were grown in 1.2 mM calcium and serum-containing medium with vitamin C (50 microg per ml) for 9 d, total lipid content remained unchanged, but both glucosylceramide and ceramide content increased. Moreover, selected ceramide and glucosylceramide species: i.e., nonhydroxy ceramide 2 and both alpha- and omega-hydroxylated sphingolipids, increased preferentially [ceramide 4 (6-hydroxy-acylceramide), ceramide 5 (alpha-hydroxyceramide), ceramide 6 (4-hydroxy-alpha-hydroxyceramide), and ceramide 7 (6-hydroxy-alpha-hydroxyceramide); and acylglucosylceramide, glucosylceramide-B, and glucosylceramide-D], whereas ceramide 1, ceramide 3, glucosylceramide-C, and sphingomyelin remained unchanged. Synthesis of the corresponding ceramide and glucosylceramide fractions was enhanced by vitamin C, attributable, in part, to increased ceramide synthase activity (over 2-fold, p = 0.01); both serine palmitoyltransferase and glucosylceramide synthase activities remained unaltered. Finally, increased vitamin C-stimulated sphingolipid production correlated with the presence of lamellar bodies with mature internal contents, an increase in covalently bound omega-hydroxyceramide, and the appearance of prominent, corneocyte-bound lipid envelopes, whereas cornified envelope formation was unchanged. Thus, in submerged human keratinocytes, vitamin C induces both increased sphingolipid production and enhancement of permeability barrier structural markers.

Regulation of sphingolipid and glycosphingolipid metabolism in extrahepatic tissues by endotoxin.

The host response to infection and inflammation is associated with multiple alterations in lipid metabolism. We have shown that endotoxin [lipopolysaccharide (LPS)] stimulates hepatic sphingolipid synthesis and increases ceramide and glucosylceramide (GlcCer) content in circulating lipoproteins in Syrian hamsters. LPS also increases the activity and mRNA levels of serine palmitoyltransferase (SPT) and GlcCer synthase, the committed enzymes in sphingolipid and glycosphingolipid (GSL) synthesis, respectively, in the liver. To determine whether sphingolipid and GSL metabolism are regulated in other tissues during the host response to infection, we examined the effect of LPS on the regulation of SPT and GlcCer synthase in extrahepatic tissues in Syrian hamsters. LPS significantly increased SPT activity in spleen and kidney after 16 h of treatment, but had no effect on SPT activity in lung and brain, suggesting that the effect of LPS on sphingolipid metabolism is tissue specific. LPS also increased SPT mRNA levels in spleen and kidney by approximately 3-fold, suggesting that the increase in SPT activity is due to an increase in SPT mRNA expression. LPS significantly increased GlcCer synthase activity in spleen and kidney, and produced 4- and 15-fold increases in GlcCer synthase mRNA levels in spleen and kidney, respectively. LPS treatment increased GlcCer content by 1.3-fold in spleen and by 6.2-fold in kidney. LPS also increased the content of ceramide trihexoside by 1.7-fold in spleen. These results suggest that LPS regulates sphingolipid and GSL metabolism in spleen and kidney. An increase in GSL metabolites in spleen and kidney during the host response to infection and inflammation may be required for modulation of immune responses and regulation of cell growth. -- Memon, R. A., W. M. Holleran, Y. Uchida, A. H. Moser, C. Grunfeld, and K. R. Feingold. Regulation of sphingolipid and glycosphingolipid metabolism in extrahepatic tissues by endotoxin. J. Lipid Res. 2001. 42: 452--459.

Epidermal sphingomyelins are precursors for selected stratum corneum ceramides.

Epidermal ceramides (Cer) comprise a heterogeneous family of seven species, including two unique omega-hydroxylated Cer, that are key components of the stratum corneum (SC) intercellular lamellar membranes responsible for the epidermal permeability barrier. Although both glucosylceramide (GlcCer) and the phospho-sphingolipid sphingomyelin (SM) are potential precursors of SC Cer, based on reported chemical structures of epidermal GlcCer and SC Cer, it is assumed that all major subfractions of SC Cer are generated from lamellar body-derived GlcCer. Yet, we and others have shown that SM-derived Cer are required for normal barrier homeostasis. Moreover, two pools of SM, one from plasma membrane, the other from lamellar body-derived contents, are potentially available for Cer production. To clarify the role of SM as a potential precursor of bulk or specific SC Cer, we compared Cer moieties in epidermal SM, Cer generated from epidermal SM by sphingomyelinase treatment, Cer within SC, and Cer that persist in Gaucher SC, where GlcCer cannot generate Cer due to an absence of beta-glucocerebrosidase. Using gas chromatography-mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance for Cer characterization, epidermal SM comprise three major subfractions with distinctive amide-linked (N-acyl) fatty acid (FA) compositions: that is, either long-chain FA (SM-1; C(22;-26)), short-chain FA (SM-2; primarily C(16)), and short-chain alpha-hydroxy FA (SM-3; C(16;-18)). In contrast, only trace quantities of omega-hydroxy FA were present. For each SM subfraction, the sphingoid base was either sphingosine or sphinganine, but phytosphingosine was not detected. Comparison of these SM with corresponding sphingomyelinase-generated epidermal Cer and SC Cer revealed that the Cer moieties of SM-1 and SM-3 are equivalent to Cer 2 (NS) and Cer 5 (AS), respectively. Moreover, both Cer 2 and Cer 5 occurred in Gaucher SC, whereas other Cer subfractions did not occur. These results indicate that two epidermal SM, that is, SM-1 and SM-3, are important precursors of two corresponding Cer in mammalian SC, that is, Cer 2 and Cer 5, but other Cer species, including the omega-hydroxy Cer species, do not derive from SM.

Permeability barrier disorder in Niemann-Pick disease: sphingomyelin-ceramide processing required for normal barrier homeostasis.

Prior studies have established the requirement for enzymatic hydrolysis of glucosylceramides to ceramide for epidermal barrier homeostasis. In this study, we asked whether sphingomyelin-derived ceramide, resulting from acid-sphingomyelinase activity, is also required for normal barrier function. We showed first, that a subset of Niemann-Pick patients with severe acid-sphingomyelinase deficiency (i.e., <2% residual activity) demonstrate abnormal permeability barrier homeostasis, i.e., delayed recovery kinetics following acute barrier disruption by cellophane tape-stripping. To obtain further mechanistic insights into the potential requirement for sphingomyelin-to-ceramide processing for the barrier, we next studied the role of acid-sphingomyelinase in hairless mouse skin. Murine epidermis contains abundant acid-sphingomyelinase activity (optimal pH 5.1-5.6). Two hours following acute barrier disruption by tape-stripping, acid-sphingomyelinase activity increases 1. 44-fold (p<0.008 versus vehicle-treated controls), an increase that is blocked by a single topical application of the acid-sphingomyelinase inhibitor, palmitoyldihydrosphingosine. Furthermore, both palmitoyldihydrosphingosine and desipramine, a chemically and mechanically unrelated acid-sphingomyelinase inhibitor, significantly delay barrier recovery both 2 and 4 h after acute barrier abrogation. Inhibitor application also causes both an increase in sphingomyelin content, and a reduction of normal extracellular lamellar membrane structures, in the stratum corneum. Both of the inhibitor-induced delays in barrier recovery can be overridden by co-applications of topical ceramide, demonstrating that an alteration of the ceramide-sphingomyelin ratio, rather than sphingomyelin accumulation, is likely responsible for the barrier abnormalities that occur with acid-sphingomyelinase deficiency. These studies demonstrate an important role for enzymatic processing of sphingomyelin-to-ceramide by acid-sphingomyelinase as a mechanism for generating a portion of the stratum corneum ceramides for permeability barrier homeostasis in mammalian skin.

Infection and inflammation induce LDL oxidation in vivo.

Epidemiological studies have shown an increased incidence of coronary artery disease in patients with chronic infections and inflammatory disorders. Because oxidative modification of lipoproteins plays a major role in atherosclerosis, the present study was designed to test the hypothesis that the host response to infection and inflammation induces lipoprotein oxidation in vivo. Lipoprotein oxidation was measured in 3 distinct models of infection and inflammation. Syrian hamsters were injected with bacterial lipopolysaccharide (LPS), zymosan, or turpentine to mimic acute infection, acute systemic inflammation, and acute localized inflammation, respectively. Levels of oxidized fatty acids in serum and lipoprotein fractions were measured by determining levels of conjugated dienes, thiobarbituric acid-reactive substances, and lipid hydroperoxides. Our results demonstrate a significant increase in conjugated dienes and thiobarbituric acid-reactive substances in serum in all 3 models. Moreover, LPS and zymosan produced a 4-fold to 6-fold increase in conjugated diene and lipid hydroperoxide levels in LDL fraction. LPS also produced a 17-fold increase in LDL content of lysophosphatidylcholine that is formed during the oxidative modification of LDL. Finally, LDL isolated from animals treated with LPS was significantly more susceptible to ex vivo oxidation with copper than LDL isolated from saline-treated animals, and a 3-fold decrease occurred in the lag phase of oxidation. These results demonstrate that the host response to infection and inflammation increases oxidized lipids in serum and induces LDL oxidation in vivo. Increased LDL oxidation during infection and inflammation may promote atherogenesis and could be a mechanism for increased incidence of coronary artery disease in patients with chronic infections and inflammatory disorders.

Omega-hydroxyceramides are required for corneocyte lipid envelope (CLE) formation and normal epidermal permeability barrier function.

Omega-hydroxyceramides (omega-OHCer) are the predominant lipid species of the corneocyte lipid envelope in the epidermis. Moreover, their omega-esterified-derivatives (acylCer) are major components of the stratum corneum extracellular lamellae, which regulate cutaneous permeability barrier function. Because epidermal omega-OHCer appear to be generated by a cytochrome P450-dependent process, we determined the effects of a mechanism-based inhibitor of omega-hydroxylation, aminobenzotriazole (ABT), on epidermal omega-OH Cer formation and barrier function. We first ascertained that ABT, but not hydroxybenzotriazole (OHBT), a chemical relative with no P450 inhibitory activity, inhibited the incorporation of [14C]-acetate into the omega-OH-containing Cer species in cultured human keratinocytes (68.1% +/- 6.9% inhibition versus vehicle-treated controls; p < 0.001), without altering the synthesis of other Cer and fatty acid species. In addition, ABT significantly inhibited the omega-hydroxylation of very long-chain fatty acids in cultured human keratinocytes. Topical application of ABT, but not OHBT, when applied to the skin of hairless mice following acute barrier disruption by tape-stripping, resulted in a significant delay in barrier recovery (e.g., 38.3% delay at 6 h versus vehicle-treated animals), assessed as increased transepidermal water loss. The ABT-induced barrier abnormality was associated with: (i) a significant decrease in the quantities of omega-OHCer in both the unbound and the covalently bound Cer pools; (ii) marked alterations of lamellar body structure and contents; and (iii) abnormal stratum corneum extracellular lamellar membrane structures, with no signs of cellular toxicity. Furthermore, pyridine-extraction of ABT- versus vehicle-treated skin, which removes all of the extracellular lamellae, leaving the covalently attached lipids, showed numerous foci with absent corneocyte lipid envelope in ABT- versus vehicle-treated stratum corneum. These results provide the first direct evidence for the importance of omega-OHCer for epidermal permeability function, and suggest further that acylCer and/or corneocyte lipid envelope are required elements in permeability barrier homeostasis.

Regulation of glycosphingolipid metabolism in liver during the acute phase response.

The host response to infection is associated with multiple alterations in lipid and lipoprotein metabolism. We have shown recently that endotoxin (lipopolysaccharide (LPS)) and cytokines enhance hepatic sphingolipid synthesis, increase the activity and mRNA levels of serine palmitoyltransferase, the first committed step in sphingolipid synthesis, and increase the content of sphingomyelin, ceramide, and glucosylceramide (GlcCer) in circulating lipoproteins in Syrian hamsters. Since the LPS-induced increase in GlcCer content of lipoproteins was far greater than that of ceramide or sphingomyelin, we have now examined the effect of LPS and cytokines on glycosphingolipid metabolism. LPS markedly increased the mRNA level of hepatic GlcCer synthase, the enzyme that catalyzes the first glycosylation step of glycosphingolipid synthesis. The LPS-induced increase in GlcCer synthase mRNA levels was seen within 2 h, sustained for 8 h, and declined to base line by 24 h. LPS-induced increase in GlcCer synthase mRNA was partly accounted for by an increase in its transcription rate. LPS produced a 3-4-fold increase in hepatic GlcCer synthase activity and significantly increased the content of GlcCer (the immediate product of GlcCer synthase reaction) as well as ceramide trihexoside and ganglioside GM3 (products distal to the GlcCer synthase step) in the liver. Moreover, both tumor necrosis factor-alpha and interleukin-1beta, cytokines that mediate many of the metabolic effects of LPS, increased hepatic GlcCer synthase mRNA levels in vivo as well as in HepG2 cells in vitro, suggesting that these cytokines can directly stimulate glycosphingolipid metabolism. These results indicate that LPS and cytokines up-regulate glycosphingolipid metabolism in vivo and in vitro. An increase in GlcCer synthase mRNA levels and activity leads to the increase in hepatic GlcCer content and may account for the increased GlcCer content in circulating lipoproteins during the acute phase response.

Beta-glucocerebrosidase activity in mammalian stratum corneum.

Although previous studies have demonstrated a crucial role for the enzyme beta-glucocerebrosidase (GlcCer'ase) in the final steps of membrane structural maturation in mammalian stratum cornuem (SC) and epidermal homeostasis, the precise in vivo localization of GlcCer'ase activity and protein is not known. Here, we developed a fluorogenic in situ assay on histologic sections (zymography) to elucidate the in vivo distribution of GlcCer'ase activity, and further characterized and localized the SC GlcCer'ase activity in vitro. The zymographic technique revealed higher GlcCer'ase activity in upper stratum granulosum and SC, both in murine and human SC; activity that was both inhibited by conduritol B epoxide, a specific GlcCer'ase inhibitor, and pH-dependent; i.e., present at pH 5.2, and absent or significantly reduced at neutral pH (7.4), consistent with the known pH optimum for epidermal GlcCer'ase in vitro. Immunohistochemical staining for GlcCer'ase protein showed enhanced fluorescent signal in the outer layers of human epidermis, concentrated at the apex and margins of stratum granulosum and lower SC. Moreover, in extracts from individual epidermal layers, GlcCer'ase activity was present throughout murine epidermis, with the highest activity in the SC, peaking in the lower-to-mid-SC. The SC activity was stimulated >10-fold by sodium taurocholate, and inhibited by bromoconduritol B epoxide. Finally, isolated membrane couplets, prepared from SC sheets, also demonstrated significant GlcCer'ase activity. These data localize GlcCer'ase activity to the outer epidermis by three different techniques, and support the role of this enzyme in extracellular processing of glucosylceramides to ceramides, required for permeability barrier maturation and function.

Sphingolipid activator proteins are required for epidermal permeability barrier formation.

The epidermal permeability barrier is maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the major components of these multilayered membranes, derive in large part from hydrolysis of glucosylceramides mediated by stratum corneum beta-glucocerebrosidase (beta-GlcCerase). Prosaposin (pSAP) is a large precursor protein that is proteolytically cleaved to form four distinct sphingolipid activator proteins, which stimulate enzymatic hydrolysis of sphingolipids, including glucosylceramide. Recently, pSAP has been eliminated in a mouse model using targeted deletion and homologous recombination. In addition to the extracutaneous findings noted previously, our present data indicate that pSAP deficiency in the epidermis has significant consequences including: 1) an accumulation of epidermal glucosylceramides together with below normal levels of ceramides; 2) alterations in lipids that are bound by ester linkages to proteins of the cornified cell envelope; 3) a thickened stratum lucidum with evidence of scaling; and 4) a striking abnormality in lamellar membrane maturation within the interstices of the stratum corneum. Together, these results demonstrate that the production of pSAP, and presumably mature sphingolipid activator protein generation, is required for normal epidermal barrier formation and function. Moreover, detection of significant amounts of covalently bound omega-OH-GlcCer in pSAP-deficient epidermis suggests that deglucosylation to omega-OH-Cer is not a requisite step prior to covalent attachment of lipid to cornified envelope proteins.

UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes.

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.

Endotoxin and cytokines increase hepatic sphingolipid biosynthesis and produce lipoproteins enriched in ceramides and sphingomyelin.

Alterations in triglyceride and cholesterol metabolism often accompany inflammatory diseases and infections. We studied the effects of endotoxin (lipopolysaccharide [LPS]) and cytokines on hepatic sphingolipid synthesis, activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid synthesis, and lipoprotein sphingolipid content in Syrian hamsters. Administration of LPS induced a 2-fold increase in hepatic SPT activity. The increase in activity first occurred at 16 hours, peaked at 24 hours, and was sustained for at least 48 hours. Low doses of LPS produced maximal increases in SPT activity, with half-maximal effect seen at approximately 0.3 microg LPS/100 g body weight. LPS increased hepatic SPT mRNA levels 2-fold, suggesting that the increase in SPT activity was due to an increase in SPT mRNA. LPS treatment also produced 75% and 2.5-fold increases in hepatic sphingomyelin and ceramide synthesis, respectively. Many of the metabolic effects of LPS are mediated by cytokines. Interleukin 1 (IL-1), but not tumor necrosis factor, increased both SPT activity and mRNA levels in the liver of intact animals, whereas both IL-1 and tumor necrosis factor increased SPT mRNA levels in HepG2 cells. IL- produced a 3-fold increase in SPT mRNA in HepG2 cells, and the half-maximal dose was 2 ng/mL. IL-1 also increased the secretion of sphingolipids into the medium. Analysis of serum lipoprotein fractions demonstrated that very low density lipoprotein, intermediate density lipoprotein, and low density lipoprotein isolated from animals treated with LPS contained significantly higher amounts of ceramide, glucosylceramide, and sphingomyelin. Taken together, these results indicate that LPS and cytokines stimulate hepatic sphingolipid synthesis, which results in an altered structure of circulating lipoproteins and may promote atherogenesis.

Barrier recovery is impeded at neutral pH, independent of ionic effects: implications for extracellular lipid processing.

Epidermal permeability barrier homeostasis requires the postsecretory processing of polar lipid precursors into nonpolar lipid products within the stratum corneum (SC) interstices by a family of lipid hydrolases. A specific requirement for beta-glucocerebrosidase (beta-GlcCer'ase), which exhibits a distinct acidic pH optimum, is particularly well documented. Therefore, we sought to determine whether the recovery of the barrier after acute insults requires acidification of the SC. We examined permeability barrier recovery by assessing changes in transepidermal water loss (TEWL), SC membrane ultrastructure utilizing ruthenium tetroxide (RuO4) postfixation, and beta-GlcCer'ase activity by in situ zymography at an acidic vs neutral pH. Barrier recovery proceeded normally when acetone-treated skin was exposed to solutions buffered to an acidic pH. In contrast, the initiation of barrier recovery was slowed when treated skin was exposed to neutral or alkaline pH, regardless of buffer composition. In addition, enhancement of the alkaline buffer-induced delay in barrier recovery occurred with Ca2+ and K+ inclusion in the buffer. Moreover, the pH-dependent alteration in barrier recovery appeared to occur through a mechanism that was independent of Ca(2+)- or K(+)-controlled lamellar body secretion, since both the formation and secretion of lamellar bodies proceeded comparably at pH 5.5 and pH 7.4. In contrast, exposure to pH 7.4 (but not pH 5.5) resulted in both the persistence of immature, extracellular lamellar membrane structures, and a marked decrease in the in situ activity of beta-GlcCer'ase. These results suggest first that an acidic extracellular pH is necessary for the initiation of barrier recovery, and second that the delay in barrier recovery is a consequence of inhibition of postsecretory lipid processing.

Glucosylceramides stimulate mitogenesis in aged murine epidermis.

Glucosylceramides (GlcCer) and ceramides (Cer) appear to have opposite effects on epidermal growth and differentiation. Whereas Cer inhibit mitosis and induce terminal differentiation and apoptosis in cultured keratinocytes, GlcCer is mitogenic in young murine epidermis. Using a recently described murine model of chronologic senescence we explored whether GlcCer is mitogenic in aged epidermis. Epidermal GlcCer content increases following topical applications of either conduritol-B epoxide (CBE), an inhibitor of GlcCer hydrolysis, or exogenous GlcCer in a penetration-enhancing vehicle. During chronologic aging in the hairless mouse, baseline epidermal DNA synthesis rates remain normal until 18 mo, but decline significantly at 24 mo. Topical CBE stimulates a 1.5- to 1.9-fold increase in epidermal DNA synthesis in all age groups (i.e., 1-2, 18, and 24 mo). Although the CBE induced increase in [3H]thymidine incorporation in 24 mo old animals is significant (p < 0.01), it is not sufficient to reach the absolute levels reached in similarly treated, younger mouse epidermis. Moreover, topical GlcCer induced mitogenesis is both dose dependent and hexose specific in young (1-2 mo old) animals, and remains effective in aged (< or = 24 mo old) animals. Furthermore, the CBE induced increase in DNA synthesis in aged epidermis is sufficient to produce epidermal hyperplasia. Finally, although an increased GlcCer:Cer ratio can alter stratum corneum barrier function and membrane structure, neither stratum corneum function nor extracellular membrane structure change under these experimental conditions, and therefore the mitogenic effects of increased epidermal GlcCer cannot be attributed to effects on the stratum corneum. These results show that: (i) elevations in endogenous GlcCer are mitogenic for aged as well as young murine epidermis; (ii) topical GlcCer is also mitogenic when delivered in an enhancing vehicle; and (iii) despite the putative importance of epidermal DNA synthesis for barrier homeostasis, these mitogenic alterations do not alter stratum corneum function.

Parallel regulation of sterol regulatory element binding protein-2 and the enzymes of cholesterol and fatty acid synthesis but not ceramide synthesis in cultured human keratinocytes and murine epidermis.

After permeability barrier perturbation there is an increase in the mRNA levels for key enzymes necessary for lipid synthesis in the epidermis. The mechanism(s) responsible for this regulation is unknown. Sterol regulatory element binding proteins-1a, 1c, and -2 (SREBPs) control the transcription of enzymes required for cholesterol and fatty acid t synthesis in response to modulations of sterol levels. We now demonstrate that SREBP-2 is the predominant SREBP in human keratinocytes and murine epidermis, while SREBP-1 is not detected. Sterols regulate SREBP-2 mRNA levels in keratinocytes and the epidermis and the proteolytic cleavage of SREBP-2 to the mature active form in keratinocytes. In parallel to the increase in mature active SREBP, there is a coordinate increase in mRNA levels for cholesterol (HMG-CoA reductase, HMG-CoA synthase, farnesyl diphosphate synthase, and squalene synthase) and fatty acid (acetyl-CoA carboxylase, fatty acid synthase) synthetic enzymes. However, mRNA levels for serine palmitoyl transferase (SPT), the first committed step for ceramide synthesis, do not increase in parallel. The increase of mRNA for enzymes required for epidermal cholesterol and fatty acid synthesis is consistent with both the previously described early increase of cholesterol and fatty acid synthesis after barrier disruption and a role for SREBP-2 in the regulation of cholesterol and fatty acid synthesis for epidermal barrier homeostasis. In contrast, SPT appears to be regulated by different mechanisms, consistent with the different time course of its stimulation after barrier disruption.

Glucosylceramide synthase activity in murine epidermis: quantitation, localization, regulation, and requirement for barrier homeostasis.

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.

Permeability barrier disruption coordinately regulates mRNA levels for key enzymes of cholesterol, fatty acid, and ceramide synthesis in the epidermis.

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.