Glucosylceramides Stimulate Mitogenesisin Aged Murine Epidermis

Nancy L. Marchell, Yoshikazu Uchida, Barbara E. Brown, Peter M. Elias, and Walter M. HolleranDermatology Service, Department of Veterans Affairs Medical Center and Department of Dermatology, School of Medicine, University of California, SanFrancisco, California, U.S.A.

Glucosylceramides (GlcCer) and ceramides (Cer) appearto have opposite effects on epidermal growth and differ-entiation. Whereas Cer inhibit mitosis and induce ter-minal differentiation and apoptosis in culturedkeratinocytes, GlcCer is mitogenic in young murineepidermis. Using a recently described murine model ofchronologic senescence we explored whether GlcCer ismitogenic in aged epidermis. Epidermal GlcCer contentincreases following topical applications of either conduri-tol-B epoxide (CBE), an inhibitor of GlcCer hydrolysis,or exogenous GlcCer in a penetration-enhancing vehicle.During chronologic aging in the hairless mouse, baselineepidermal DNA synthesis rates remain normal until18 mo, but decline significantly at 24 mo. Topical CBEstimulates a 1.5- to 1.9-fold increase in epidermal DNAsynthesis in all age groups (i.e., 1–2, 18, and 24 mo).Although the CBE induced increase in [3H]thymidineincorporation in 24 mo old animals is significant(p , 0.01), it is not sufficient to reach the absolute levelsreached in similarly treated, younger mouse epidermis.

Sphingolipids are emerging as a novel family of lipid secondmessengers, which mediate diverse effects on growth, differ-entiation, and apoptosis in several cell types and tissues.Whereas sphingoid bases, related lysosphingolipid species,and ceramides (Cer) generally (reviewed in Kolesnick, 1991;

Hannun, 1996), but not always (Olivera et al, 1992), inhibit mitosisand induce differentiation, other sphingoid base metabolites, such asglucosylceramides (GlcCer) (Datta and Radin, 1988; Shayman et al,1991; Marsh et al, 1995), lysosphingomyelin (Desai and Spiegel, 1991),lactosylceramide (Chatterjee, 1991), and more complex glycosphingo-lipids (Tsuji et al, 1983; Katoh-Semba et al, 1986; Hanai et al, 1988),can stimulate growth. For example, the organomegaly in Gaucherdisease reflects not only tissue accumulation of GlcCer (Barranger andGinns, 1989), but also putative growth-stimulatory effects of GlcCer(Datta and Radin, 1988). Moreover, the kidney hypertrophy thatoccurs in experimental diabetes is reversed by inhibitors of GlcCer

Manuscript received June 17, 1997; revised October 16, 1997; accepted forpublication November 24, 1997.

Reprint requests to: Dr. Walter M. Holleran, Dermatology Service (190),Veterans Administration Medical Center, 4150 Clement Street, San Francisco,California 94121.

Abbreviations: CBE, conduritol-B epoxide; Cer, ceramide; GalCer, β-D-galactosylceramide; GlcCer, β-D-glucosylceramide; TEWL, transepidermalwater loss.

0022-202X/98/$10.50 · Copyright © 1998 by The Society for Investigative Dermatology, Inc.

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Moreover, topical GlcCer induced mitogenesis is bothdose dependent and hexose specific in young (1–2 moold) animals, and remains effective in aged (ø24 mo old)animals. Furthermore, the CBE induced increase in DNAsynthesis in aged epidermis is sufficient to produce epi-dermal hyperplasia. Finally, although an increasedGlcCer:Cer ratio can alter stratum corneum barrier func-tion and membrane structure, neither stratum corneumfunction nor extracellular membrane structure changeunder these experimental conditions, and therefore themitogenic effects of increased epidermal GlcCer cannotbe attributed to effects on the stratum corneum. Theseresults show that: (i) elevations in endogenous GlcCerare mitogenic for aged as well as young murine epidermis;(ii) topical GlcCer is also mitogenic when deliveredin an enhancing vehicle; and (iii) despite the putativeimportance of epidermal DNA synthesis for barrierhomeostasis, these mitogenic alterations do not alterstratum corneum function. Key words: aging/ceramides/epidermal DNA synthesis/Gaucher disease. J Invest Dermatol110:383–387, 1998

synthase in association with declining tissue levels of GlcCer (Zadoret al, 1993). In addition, experimentally induced increases in GlcCerby either inhibition of GlcCer hydrolysis (Hara and Radin, 1979;Shayman et al, 1991) or direct injection of GlcCer (Datta and Radin,1988), enhance tissue proliferation. We recently showed similarlythat either inhibition of β-glucocerebrosidase with topical conduritolcompounds, or intracutaneous administration of exogenous GlcCer,results in epidermal hyperproliferation in young hairless mice (Marshet al, 1995). Furthermore, the growth stimulatory properties of theβ-glucocerebrosidase inhibitors correlate with accumulation of GlcCerin the mitotically active, basal layers of the epidermis, and areindependent of effects on barrier function or initiation of inflammation(Marsh et al, 1995). These studies suggest that GlcCer and Cer performopposite growth and differentiation related functions in the epidermis,with the tissue normally maintaining a balance in these two sphingo-lipids.

Skin aging is accompanied by well-characterized abnormalities inthe dermis, e.g., photoaging associated collagen and proteoglycanproteolysis with elastosis (Balin and Kligman, 1989a). In contrast, littleis known about changes in the epidermis with chronologic aging.Although epidermal proliferation declines with chronologic aging(Balin and Kligman, 1989b; Cerimele et al, 1990; Haratake et al, 1997),the biochemical and molecular basis for this change is not known. Inaddition, the epidermal permeability barrier is also compromised withadvanced chronologic aging (Ghadially et al, 1995), an abnormality

384 MARCHELL ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

attributed to the defective generation of epidermal lipids (Ghadiallyet al, 1995). It is possible that reduced epidermal growth rates contributeto declining lipid generation and deposition, because mitogens such asalpha-hydroxyacids reverse many features of chronologic and photo-aging. In this study, we asked whether modulation of epidermal GlcCercontent with topical administration of either conduritol inhibitors orGlcCer would enhance epidermal growth in aged epidermis. Ourstudies show that both approaches stimulate epidermal growth in agedepidermis, suggesting that reduced GlcCer could contribute to thefunctional alterations in cutaneous aging.

MATERIALS AND METHODS

Materials Glucocerebrosides (Gaucher spleen) were obtained from Matreya(Philadelphia, PA); conduritol B epoxide (CBE) was from Toronto ResearchChemicals (Toronto, Canada). Calf thymus DNA was obtained from Sigma(St. Louis, MO), dithiothreitol was obtained from Boehringer (Indianapolis,IN), and [methyl-3H] thymidine was from Amersham Life Sciences (ArlingtonHeights, IL). All solvents were reagent or high performance liquid chromato-graphy grade.

Animals and experimental protocols Male hairless mice (h/h) from CharlesRiver (Hollister, CA) were used in these studies, because these animals liveto ù 24 mo of age without premature development of tumors (Ghadially et al,1995), as occurs with other commercially available hairless murine strains.Cohorts of mice were studied between 1 and 2, 18, or 24 mo of age, becauseprior studies showed that abnormal epidermal function does not occur in theseanimals until ù 18 mo (Ghadially et al, 1995). Animals were treated with either(i) a single, daily topical application of CBE (100 µg) or (ii) monohexosyl-ceramides [GlcCer or galactosylCer (GalCer)], 2.5 mg twice daily in a propyleneglycol:ethanol (PG:EtOH; 7:3 vols) or a penetration-enhancing vehicle (CellegyPharmaceuticals, San Carlos, CA) applied to a 2.5 cm2 area of one flank. Theopposite flank was left either untreated or was treated with vehicle alone.Additional groups of animals were treated with GlcCer at doses of 0.5, 1.0,2.0, or 2.5 mg twice daily in the penetration-enhancing vehicle, which provedto be more effective than PG:EtOH.

Epidermal DNA synthesis Twenty-four hours after the initial treatment,animals were sacrificed and flank skin removed. Both treated and control skinsamples were scraped free of excess subcutaneous fat, and 6 mm punch sampleswere placed into 1.0 ml of serum-free, keratinocyte growth medium (Clonetics,San Diego, CA). One microcurie of [3H]thymidine was added to each well,and tissue samples were incubated at 37°C for 2 h. Samples were rinsed threetimes with phosphate-buffered saline, homogenized in phosphate-bufferedsaline, and an equal volume of cold trichloroacetic acid (20% by weight) wasadded. The pellet was rinsed twice with cold 5% trichloroacetic acid, resuspendedin 1N NaOH, and aliquots were counted by liquid scintillation spectrometry.Total cellular DNA was assayed by the method of Labarca and Paigan (1980).Samples for DNA assay were brought to 1 ml with Hoechst buffer (pH 7.4).One milliliter of bisbenzimidazole (Sigma; diluted 1:1000 with distilled H2O)was added. Standards were prepared using calf thymus DNA (137 µg per ml)diluted 1:10 with Hoechst buffer. All samples and standards were kept in thedark for 2 h, and fluorescence was measured using a spectrophotometer.Statistical differences were calculated using a Student’s two-tailed t test.

Histology Fresh, full-thickness skin samples were obtained before treatment,and at day 5, following alternate-day treatment (i.e., days 1, 3, and 5) withCBE or vehicle. The alternate-day regimen was used to accentuate the histologicevidence for hyperplasia (subsequent to the induced hyperproliferation at 24 hfollowing a single application), without evidence for deleterious effects onbarrier integrity [i.e., no change in transepidermal water loss (TEWL) occurwith this regimen]. Biopsies were taken for light microscopy (hematoxylin andeosin stain), fixed in buffered formalin, paraffin-embedded, and sectioned(5 µm). All sections were examined and photographed with a Leitz OrtholuxII microscope.

Assessment of stratum corneum function Cohorts of aged (24 mo) andyoung (1–2 mo) mice were treated with either the GlcCer in vehicle or vehiclealone twice daily, on days 1, 3, and 5 as above. Immediately after the lastapplication of lipid versus vehicle, the stratum corneum of treated and untreatedsites was stripped successively with cellophane tape until TEWL rates exceeded6 mg per cm2 per h. The number of strippings required to reach this level ofbarrier abrogation was considered an indicator of stratum corneum integrity(5 cohesion). After tape-stripping, barrier function was measured at regularintervals until function had normalized, utilizing an electrolytic water analyzer(Meeco, Warrington, PA), as described previously (Holleran et al, 1991). Waterloss measurements were obtained over a small area of skin (0.5 cm2), recorded

Figure 1. Stimulation of [3H]thymidine incorporation by topical CBEin young versus aging epidermis. Twenty-four hours following initialtreatment with CBE, whole skin punch biopsy samples were incubated with[3H]thymidine in organ culture (see Materials and Methods). Baseline (vehicletreated) incorporation was significantly diminished in 24 mo old animals(*p , 0.01 versus either 1–2 or 18 mo old animals). Increased incorporationwas observed in all animals treated with CBE regardless of age [significance forCBE versus vehicle treated samples for each age group: a, p , 0.005 (n 5 12);b, p , 0.001 (n 5 25); and c, p , 0.01 (n 5 6), for 1–2, 18, and . 24 moold animals, respectively]. Results are presented as mean [3H]-incorporateddisintegrations per min (DPM) per cm2 6 SEM.

in parts per million per 0.5 cm2 per h over background, and the data convertedto mg per cm2 per h. At each time point, 3–5 readings were performed oneach flank (treated, vehicle treated, and untreated), with 6–8 animals in eachtest group.

Electron microscopy In order to assess whether the mitogenic response ofaged epidermis to CBE or GlcCer could be attributed to stimulation of DNAsynthesis secondary to altered barrier function, we obtained full thickness skinbiopsies for electron microscopy at 24 h and at day 5 following alternate-daytreatments (i.e., days 1, 3, and 5, as above). Samples were fixed in half-strengthKarnovsky’s fixative, divided, and processed through reduced 1.0% osmiumtetroxide or 0.25% ruthenium tetroxide (Hou et al, 1991), followed byembedding in an Epon-epoxy mixture (McNutt and Crain, 1981). Full-thickness biopsies from the flanks of both aged (. 18 mo) and young (6–8 wkold) hairless mice were also processed, as described above. Ultrathin sectionswere viewed in an electron microscope (10 A; Carl Zeiss, Thornwood, NY)after further contrasting in lead citrate and uranyl acetate.

RESULTS

Inhibition of β-glucocerebrosidase stimulates mitogenesis inaged mouse epidermis Inhibition of epidermal β-glucocerebrosid-ase with CBE is accompanied by increased DNA synthesis andepidermal hyperplasia, with localized accumulation of GlcCer in youngmurine epidermis (Marsh et al, 1995). Therefore, we first assessedwhether aged epidermis would display a comparable response to topicalCBE treatment. As seen in Fig 1, young (1–2 mo) murine epidermisdisplays a 50% increase (i.e., 1.5-fold) in DNA synthesis after twotopical applications of CBE versus vehicle (p , 0.005). Moreover,18 mo old epidermis also displays rates of DNA synthesis that are stillcomparable with young epidermis in vehicle treated sites, and theepidermis generates a 90% increase in DNA synthesis with CBE versusvehicle treatment (p , 0.001). By 24 mo, basal rates of epidermal DNAsynthesis are depressed significantly (50% reduction) in comparison withrates in young and 18 mo old skin (p , 0.01). Nevertheless, 24 moold epidermis also responds to CBE treatment with a 90% increase inDNA synthesis; however, the increase at 24 mo is not sufficient toattain the absolute rates reached in 1–2 or 18 mo epidermis treatedwith CBE. These results confirm the mitogenic response to CBE inyoung murine epidermis, show that epidermal DNA synthesis doesnot decline until after 18 mo in murine skin, and show that agedepidermis generates a mitogenic response comparable with youngepidermis, but that the response in 24 mo skin does not attain levelscomparable with those in younger epidermis.

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Figure 2. Stimulation of [3H]thymidine incorporation by topical GlcCerin young (1–2 mo old) murine epidermis. Twenty-four hours followingtopical application of vehicle, penetration-enhancing vehicle (enhancer), orenhancer plus either GlcCer or GalCer (each 2.5 mg), punch biopsy sampleswere incubated in organ culture with [3H]thymidine as described (see Materialsand Methods). Topical GlcCer treatment significantly increased incorporationover vehicle or enhancer treated controls in young epidermis (p , 0.001; n 56); topical GalCer did not alter incorporation relative to controls. Thepenetration-enhancing vehicle (see Materials and Methods) did not significantlyalter baseline rate of incorporation. Results presented as mean DPM percm2 6 SEM.

Figure 3. Mitogenic effect of topical GlcCer is dose dependent. Increasingamounts of topical GlcCer (0.5–2.5 mg) were applied to flanks of young (1–2 mo) mice. Punch biopsies and thymidine incorporation were performed asdescribed above. Increasing amounts of topical GlcCer corresponded withincreased stimulation of epidermal mitogenesis (thymidine incorporation) 24 hfollowing initial application. *For 2.5 mg GlcCer dose, p , 0.005 (n 5 4animals) versus vehicle-enhancer control (n 5 14); values are mean 6 SD.Lower GlcCer doses (0.5, 1.0, 2.0 mg) were performed on duplicate animalsonly (n 5 2), and reported as average 6 range/2.

Topical glucosylceramides stimulate mitogenesis in young andaged mouse epidermis Intracutaneous injections of GlcCer (butnot other cerebrosides) were shown to stimulate DNA synthesis inyoung murine epidermis (Marsh et al, 1995). Because topical applicationsof GlcCer in a standard, PG:EtOH vehicle were not effective inmimicking this effect (not shown), we next ascertained whether GlcCerwould stimulate mitogenesis, if applied from a penetration-enhancingvehicle expected to improve penetration through the stratum corneum.As shown in Fig 2, topical GlcCer stimulated an approximately 2-foldincrease in epidermal DNA synthesis in comparison with both theenhancing and the standard vehicles. In addition, the effect of thesphingolipid is hexose specific, i.e., as described previously in youngmurine skin for intracutaneous GalCer (Marsh et al, 1995), no responseoccurs with topical GalCer in young murine skin (Fig 2). Moreover,the highest response (2.0-fold increase) occurs at a final dose of 2.5 mg,whereas lower doses produced lesser increases in DNA synthesis (Fig 3).

Figure 4. Topical GlcCer stimulates [3H]thymidine incorporation inaged (24 mo) epidermis. Topical GlcCer (2.5 mg) was applied to the backof either young (1–2 mo) or aged (. 24 mo old) mice as before (Fig 2).Whole skin punch biopsy samples were taken 24 h following the initialapplication of penetration-enhancing vehicle (control), or enhancer plus GlcCer(2.5 mg), incubated in organ culture with [3H]thymidine. Both young (1.5-fold) and aged (1.6-fold) murine epidermis demonstrated significantly enhancedthymidine incorporation over vehicle treated control samples (p , 0.005 foreach; n 5 5 animals). Values are mean 6 SD.

We next determined whether topical GlcCer application wouldresult in an increased mitogenic response in aged animal epidermis.Topical GlcCer induces a significant increase in epidermal mitogenesisboth in young (1–2 mo old) and in aged (. 24 wk old) murineepidermis (Fig 4). Moreover, despite the lower baseline mitogenicrate in aged animal epidermis, the fold-increase in [3H]thymidineincorporation is equivalent for young and aged animals (i.e., 1.5- to1.6-fold), and comparable with the increase induced with CBE in agedskin. These results show that topical GlcCer is able to stimulateepidermal DNA synthesis, in a concentration, vehicle, and hexosedependent fashion, regardless of age.

Inhibition of β-glucocerebrosidase leads to hyperplasia of agedmurine epidermis Because topical conduritol compounds produceboth increased epidermal DNA synthesis and epidermal hyperplasia inyoung murine skin (compare with Fig 1; Marsh et al, 1995), wedetermined whether CBE produced a similar hyperplasia response inaged animals. Following twice-daily applications of CBE on alternatedays for 5 d, aged epidermis displays an increased number of cell layersin comparison with adjacent vehicle treated sites (Fig 5). The alternate-day regimen was used to accentuate hyperplasia; no change in TEWLwas evident with this regimen. Moreover, there is no evidence ofinflammatory cells in the dermis following CBE treatment, supportingearlier studies that CBE induced epidermal hyperplasia cannot beattributed to nonspecific irritant effects (Marsh et al, 1995).

Prior studies have also shown both that barrier function regulatesepidermal DNA synthesis (Proksch et al, 1991) and that chronic, single-daily treatment (topical BrCBE for . 5 d; Holleran et al, 1993, 1994),or chronic, twice-daily treatment (CBE for . 3 d; Marsh et al, 1995),can abrogate epidermal barrier function. The above studies show thatalternate-day CBE treatment (i.e., days 1, 3, and 5) does not alterbarrier function (no change in TEWL) in aged epidermis. Using thismodified regimen, we next determined whether CBE treatmentalters stratum corneum membrane structure, as a further, independentindicator of barrier dysfunction. Neither CBE nor vehicle treatmentmodified membrane structure in aged stratum corneum during thecourse of these studies (not shown). Although the overall content ofintercellular lamellae is decreased in both groups, as described previouslyfor aged murine stratum corneum (Ghadially et al, 1995), normallamellar unit structures predominate in both groups (not shown). Thesestudies, together with the TEWL assays above, show that CBE modifiesneither stratum corneum structure nor function during the course ofthese studies.

386 MARCHELL ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 5. Histology of CBE treated aged (24 mo old) epidermis.Hematoxylin and eosin stain of vehicle treated (A) and CBE treated (B) agedmurine skin. Skin was treated with topical CBE on alternate days (i.e., day 1,3, and 5) (see Materials and Methods). Hyperplasia is evident in CBE treatedepidermis (B), whereas vehicle treated controls appear normal (A). · · · · ·,dermal–epidermal junction; d, dermis; magnification, 3 150; scale bar, 20 µm.

CBE induced enhancement of epidermal DNA synthesis doesnot lead to improvement in stratum corneum function Priorstudies have shown that barrier function is an important regulator ofepidermal DNA synthesis (Proksch et al, 1991); however, whether theenhanced DNA synthesis after acute insults is required for barrierrecovery is not known. Because aged murine epidermis displays bothdefective stratum corneum cohesion and barrier recovery (Ghadiallyet al, 1995), we next examined whether the CBE induced enhancementof epidermal DNA synthesis improves stratum corneum function.Cohorts of 24 mo old mice were treated (days 1, 3, and 5) with eitherCBE or vehicle, conditions that increase DNA synthesis about 2-fold(compare with Fig 1). On the fifth day, baseline stratum corneumbarrier function was measured, followed by sequential tape strippingas a measure of integrity, and TEWL was measured again at severaltime points after tape stripping (barrier recovery). This CBE treatmentregimen did not result in altered barrier function in aged epidermis(i.e., no change from baseline; not shown), similar to that reportedpreviously for young murine skin following 1–3 d of daily CBE(Holleran et al, 1993; Marsh et al, 1995). Moreover, the number ofstrippings required to reach a TEWL ù 8 mg per cm2 per h wasnot significantly different in the CBE versus vehicle treated groups(approximately four strippings in each group). Finally, the kinetics ofbarrier recovery were comparable in the CBE versus vehicle treated,aged animals (not shown).

DISCUSSION

In the stratum corneum of terrestrial mammals, ceramides are generatedfrom GlcCer precursors by the activity of β-glucocerebrosidase (Wertzand Downing, 1989; Holleran et al, 1992), a process critical for normal

permeability barrrier homeostasis (Holleran et al, 1993, 1994); however,GlcCer also appear to have other important, independent regulatoryroles in epidermal homeostasis. For example, we demonstrated previ-ously that increased epidermal GlcCer induces hyperplasia in youngmurine epidermis (Marsh et al, 1995). Studies in cultured keratinocyteshave also shown that GlcCer synthase inhibitors slow growth (Wakitaet al, 1994)1, whereas β-glucocerebrosidase inhibitors stimulate growth;however, excess GlcCer is readily hydrolyzed to Cer, to restore basalgrowth kinetics to cultured keratinocytes.1 This study demonstratesthat aged murine epidermis retains its sensitivity to the mitogeniceffects of GlcCer, providing further evidence that sphingolipids canhave additional effects in the regulation of epidermal homeostasis.

Chronologic aging of mammalian skin is associated with severalalterations, including a well-known decrease in epidermal turnover(Balin and Kligman, 1989b; Cerimele et al, 1990). Aged murineepidermis not only demonstrates a diminished baseline [3H]thymidineincorporation and epidermal thickness, but also an attenuated mitoticresponse to high-dose UVB exposure (Haratake et al, 1997). In thisstudy, aged murine epidermis (i.e., . 24 wk old), despite its lowerbaseline epidermal proliferation rates, retains its responsiveness to thepro-mitogenic effects of increased epidermal GlcCer. Both endogenousmanipulation of GlcCer (i.e., CBE inhibition of GlcCer hydrolysis)and exogenous application of GlcCer (i.e., subcutaneous injection ortopical application) results in a nearly equivalent increase in epidermalproliferation. The epidermal GlcCer response was also found to bedose dependent (this study), specific for the glucose-containing Cer (i.e.,galactosyl-Cer does not show mitogenic effects), and a consequenceof neither inflammation nor permeability barrier disruption (Marshet al, 1995).

The physiologic role of GlcCer in the epidermis and other tissuesremains unresolved, as does their mechanism of action. Nevertheless,the results presented here and previously (Marsh et al, 1995) suggestthat cellular GlcCer levels are at least permissive in the regulation ofepidermal proliferation. These results also suggest that the ichthyoticskin findings in both human and murine β-glucocerebrosidase defi-ciency may result, in part, from excessive GlcCer in the epidermis(Holleran et al, 1994; Sidransky et al, 1996). Our results mirrorcomparable studies in other Gaucher tissues, where the lack ofβ-glucocerebrosidase also results in abnormally elevated GlcCer levels inassociation with organomegaly (Barranger and Ginns, 1989). Moreover,pharmacologic manipulations of cellular and tissue GlcCer levels inthe whole animal also appear to mimic the organomegaly associatedwith Gaucher disease (Hara and Radin, 1979; Datta and Radin, 1988;Holleran et al, 1994). Whether high turnover, actively dividing cells,in general, exhibit higher levels of GlcCer relative to quiescent cellshas not yet been addressed; however, inhibition of GlcCer synthesishas been associated with anti-tumor activity (Inokuchi et al, 1987).Furthermore, elevated cellular GlcCer has recently been correlatedwith multidrug resistance in cancer cells (Lavie et al, 1996), andtreatment of resistant MCF-7 human breast cancer cells with theGlcCer synthase inhibitor, PDMP, restores sensitivity to adriamycin(Lavie et al, 1997); however, despite the elevated serum and tissueGlcCer levels in Gaucher patients, a higher risk of aggressive canceror metastasis has not been reported for this patient population.

An increasing variety of regulatory effects has been attributedto other glycosylated and nonglycosylated sphingolipid metabolites.Exogenously supplied Cer, in general, inhibit cellular proliferation andinduce differentiation and/or apoptosis (Okazaki et al, 1990; Kolesnick,1991; reviewed in Hannun, 1996). Likewise, proliferation of culturedkeratinocytes is inhibited similarly (Wakita et al, 1994).1 Conversely,and in accordance with this study, increased GlcCer accelerates thegrowth of cultured human keratinocytes; however, at higher GlcCerconcentrations, Cer accumulates due to GlcCer hydrolysis, leading tosignificant inhibition of keratinocyte proliferation.1 Taken together,

1Uchida Y, Schmuth M, Elias PM, Holleran WM: Divergent effects ofceramides and glucosylceramides on human keratinocyte proliferation.Submitted.

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these results suggest that GlcCer and Cer may have opposing roles inthe regulation of cellular proliferation.

Lactosylceramide, which contains the GlcCer core structure withan additional galactose residue, stimulates aortic smooth muscle cellproliferation (Chatterjee, 1991), an effect that appears to involve MAPkinase activation (Bhunia et al, 1996). Furthermore, the stimulatoryeffects of GlcCer in proximal tubular cells of polycystic kidney diseasehave been attributed to conversion of GlcCer to lactosyleramide by aspecific galactosyltransferase (Chatterjee et al, 1996). Whether theeffects of GlcCer on intact murine epidermis described here requireprior conversion to lactosylceramide has yet to be determined.

Finally, the N-deacylated metabolite of sphingomyelin, or sphingos-ylphosphorylcholine, is not only elevated in the stratum corneum ofatopic dermatitis patients (Murata et al, 1996), but also has pro-mitogenic effects in several cell types (Desai and Spiegel, 1991),including dermal 3T3 fibroblasts and endothelial cells (Sun et al, 1996).In addition, in healing-impaired diabetic mice, sphingosylphosphoryl-choline treatment improves wound healing and increases mitoticactivity in cells of the basal epidermal layer (Sun et al, 1996). Althoughthe mechanism by which sphingosylphosphorylcholine effects cellularactivities has not been fully delineated, calcium mobilization andarachidonic acid release appear to be involved (Ghosh et al, 1990;Desai et al, 1993; Kindman et al, 1994).

In summary, we have shown that modulation of epidermal GlcCercontent with either β-glucocerebrosidase inhibitors, such as CBE, orwith GlcCer itself can enhance epidermal growth in both young andaged epidermis. These results suggest that manipulation of the levelsof epidermal ceramide metabolites may be a novel approach toregulating epidermal turnover and homeostasis in appropriate diseasestates.

These studies were supported by NIH grants AR 19098, AR 39448 [PP], and theMedical Research Service, Veterans Administration. Ms. Debra Crumrine and Dr. WenNi Gao provided excellent technical assistance. Ms. Sue Allen expertly preparedthis manuscript.

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