Effect of the Lactic Acid BacteriumStreptococcus thermophilus on Ceramide Levels in Human KeratinocytesIn Vitro and Stratum Corneum In Vivo
1999; Elsevier BV; Volume: 113; Issue: 1 Linguagem: Inglês
10.1046/j.1523-1747.1999.00633.x
ISSN1523-1747
AutoresLuisa Di Marzio, Benedetta Cinque, Clara De Simone, Maria Grazia Cifone,
Tópico(s)Sphingolipid Metabolism and Signaling
ResumoThe effects of Streptococcus thermophilus on ceramide levels either in vitro on cultured human keratinocytes or in vivo on stratum corneum, have been investigated. In vitro, Streptococcus thermophilus enhanced the levels of ceramides in keratinocytes in a time-dependent way. The presence of high levels of neutral, glutathione-sensitive, sphingomyelinase in Streptococcus thermophilus could be responsible for the observed ceramide increase. The application of a base cream containing sonicated Streptococcus thermophilus in the forearm skin of 17 healthy volunteers for 7 d also led to a significant and relevant increase of skin ceramide amounts, which could be due to the sphingomyelin hydrolysis through bacterial neutral sphingomyelinase. Indeed, similar results were obtained with a base cream containing purified bacterial neutral sphingomyelinase. In addition, the inhibition of bacterial neutral sphingomyelinase activity through glutathione blocked the skin ceramide increase observed after the treatment. The topical application of a sonicated Streptococcus thermophilus preparation, leading to increased stratum corneum ceramide levels, could thus result in the improvement of lipid barrier and a more effective resistance against xerosis. The effects of Streptococcus thermophilus on ceramide levels either in vitro on cultured human keratinocytes or in vivo on stratum corneum, have been investigated. In vitro, Streptococcus thermophilus enhanced the levels of ceramides in keratinocytes in a time-dependent way. The presence of high levels of neutral, glutathione-sensitive, sphingomyelinase in Streptococcus thermophilus could be responsible for the observed ceramide increase. The application of a base cream containing sonicated Streptococcus thermophilus in the forearm skin of 17 healthy volunteers for 7 d also led to a significant and relevant increase of skin ceramide amounts, which could be due to the sphingomyelin hydrolysis through bacterial neutral sphingomyelinase. Indeed, similar results were obtained with a base cream containing purified bacterial neutral sphingomyelinase. In addition, the inhibition of bacterial neutral sphingomyelinase activity through glutathione blocked the skin ceramide increase observed after the treatment. The topical application of a sonicated Streptococcus thermophilus preparation, leading to increased stratum corneum ceramide levels, could thus result in the improvement of lipid barrier and a more effective resistance against xerosis. acidic sphingomyelinase diacylglycerol glutathione neutral sphingomyelinase phosphatidylcholine sphingomyelin thin-layer chromatography The normal morphology of stratum corneum is essential for maintaining the water barrier of the skin (Bowser and White, 1985Bowser P. White R. Isolation barrier properties and lipid analysis of the stratum compectum—a discrete region of the stratum corneum.J Invest Dermatol. 1985; 112: 1-14Google Scholar;Rawlings et al., 1994Rawlings A.V. Scott I.R. Harding C.R. Bowser P. Stratum corneum moisturization at the molecular level.J Invest Dermatol. 1994; 103: 731-740Crossref PubMed Scopus (385) Google Scholar). The protein-enriched corneocytes are embedded in an intercellular lipid matrix which is composed primarily of ceramides (43–46% of total lipids), cholesterol, and fatty acids together with smaller amounts of cholesterol sulfate, glucosylceramides, and phospholipids (Yardley and Summerly, 1981Yardley H.J. Summerly R. Lipid composition and metabolism in normal and diseased epidermis.Pharmacol Ther. 1981; 13: 357-383Crossref PubMed Scopus (198) Google Scholar;Elias et al., 1988Elias P.M. Menon G.K. Grayson S. Brown B.E. Membrane structural alterations in murine stratum corneum. Relationship to the localization of polar lipids and phospholipases.J Invest Dermatol. 1988; 91: 3-10Crossref PubMed Scopus (96) Google Scholar). These lipids form multilamellar sheets within the intercellular spaces of the stratum corneum, the organization of which is essential in maintaining the functionality of the skin as an effective barrier to water loss (Rougier et al., 1983Rougier A. Dupuis D. Lotte C. Roguet R. Schaffer H. In vivo correlation between stratum corneum reservoir function and percutaneous absorption.J Invest Dermatol. 1983; 81: 275-278Abstract Full Text PDF PubMed Scopus (159) Google Scholar;Eckert and Rorke, 1989Eckert R.L. Rorke E.A. Molecular biology of keratinocyte differentiation.Environ Health Perspect. 1989; 80: 109-116Crossref PubMed Scopus (110) Google Scholar;Potts and Francoeur, 1991Potts R.O. Francoeur M.L. The influence of stratum corneum morphology on water permeability.J Invest Dermatol. 1991; 96: 495-499Abstract Full Text PDF PubMed Google Scholar). In addition, they also play an important part in determining the mechanical, cohesive, and desquamatory properties of the stratum corneum, and therefore have a key role to counteract the environmental challenges which can lead to disturbances in skin function (for a review seeRawlings et al., 1994Rawlings A.V. Scott I.R. Harding C.R. Bowser P. Stratum corneum moisturization at the molecular level.J Invest Dermatol. 1994; 103: 731-740Crossref PubMed Scopus (385) Google Scholar). A global decrease in lipid content leads to alterations in lamellar bilayer morphology which appear to underlie the impaired functional abnormalities commonly associated with aging. Indeed, an age-related decline in stratum corneum ceramide, cholesterol, and fatty acid levels has been previously reported (Ghadially et al., 1995Ghadially R. Brown B. Sequeira-Martin S. Feingold K. Elias P. The aged epidermal permeability barrier.J Clin Invest. 1995; 95: 2281-2290Crossref PubMed Scopus (400) Google Scholar;Rogers et al., 1996Rogers J. Harding C. Mayo A. Banks J. Rawlings A.V. Stratum corneum lipids: the effect of ageing and the seasons.Arch Dermatol Res. 1996; 288: 765-770Crossref PubMed Scopus (291) Google Scholar). Overall, the total lipid levels decrease by approximately 30%, which may reflect the slower keratinocyte metabolism of the aged (Grove and Kligman, 1983Grove G.L. Kligman A.M. Age-associated change in human epidermal cell renewal.J Gerontol. 1983; 38: 137-142Crossref PubMed Scopus (156) Google Scholar), leading to decreased biosynthetic capacity. Recently, a study demonstrated a seasonal-related reduction in stratum corneum lipid levels probably reflecting decreased epidermal lipid biosynthesis (Rogers et al., 1996Rogers J. Harding C. Mayo A. Banks J. Rawlings A.V. Stratum corneum lipids: the effect of ageing and the seasons.Arch Dermatol Res. 1996; 288: 765-770Crossref PubMed Scopus (291) Google Scholar). Apart from the increased susceptibility of the barrier to damage in the elderly, the reduction in lipid levels could explain the increased incidence of xerosis in winter (Rogers et al., 1996Rogers J. Harding C. Mayo A. Banks J. Rawlings A.V. Stratum corneum lipids: the effect of ageing and the seasons.Arch Dermatol Res. 1996; 288: 765-770Crossref PubMed Scopus (291) Google Scholar). The reduction in lipid levels may in turn reduce the water content of the stratum corneum. This may influence the activity of the stratum corneum proteases thought to be involved in desquamation (Lundstrom and Egullrud, 1990Lundstrom A. Egullrud T. Cell shedding from human plantar skin in vitro: evidence that two different types of protein structures are degraded by a chimotrypsin-like enzyme.Arch Dermatol Res. 1990; 282: 234-237Crossref PubMed Scopus (47) Google Scholar;Egelrud, 1993Egelrud T. Purification and preliminary characterization of stratum corneum chymotryptic enzyme: a proteinase that may be involved in desquamation.J Invest Dermatol. 1993; 101: 200-204Abstract Full Text PDF PubMed Google Scholar;Rawlings et al., 1994Rawlings A.V. Scott I.R. Harding C.R. Bowser P. Stratum corneum moisturization at the molecular level.J Invest Dermatol. 1994; 103: 731-740Crossref PubMed Scopus (385) Google Scholar) and interferes with the generation of natural moisturizing factors (Scott and Harding, 1986Scott I. Harding C. Fillagrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment.Dev Biol. 1986; 115: 84-92Crossref PubMed Scopus (256) Google Scholar), leaving the stratum corneum more susceptible to xerosis. The reduction of the stratum corneum ceramide levels have been proposed as a possible etiologic factor in atopic dermatitis and psoriasis (Motta et al., 1994Motta S. Monti M. Sesana S. Mellesi L. Ghidoni R. Caputo R. Abnormality of water barrier function in psoriasis.Arch Dermatol. 1994; 130: 452-456Crossref PubMed Scopus (204) Google Scholar;Murata et al., 1996Murata Y. Ogata J. Higaki Y. et al.Abnormal expression of sphingomyelin acylase in atopic dermatitis: an etiologic factor for ceramide deficiency?.J Invest Dermatol. 1996; 106: 1242-1249Crossref PubMed Scopus (193) Google Scholar). Moreover, topical application of ceramides has been shown to improve directly the barrier function of the stratum corneum (Imokawa et al., 1989Imokawa G. Akasaki S. Minematsu Y. Kaway M. Importance of intercellular lipids in water retention properties of the stratum corneum: induction and recovery study of surfactant dry skin.Arch Dermatol Res. 1989; 281: 45-51Crossref PubMed Scopus (205) Google Scholar). Epidermis possesses the capacity to synthesize all lipids required for the barrier formation. Especially unique for the epidermis is the synthesis of large amounts of glucosylceramides and ceramides (Heldberg et al., 1988Heldberg C.L. Werts P.W. Downing D.T. The time course of lipid biosynthesis in pig epidermis.J Invest Dermatol. 1988; 91: 169-174Crossref Scopus (52) Google Scholar), which has also been demonstrated in organotypic keratinocyte cultures (Schurer et al., 1989Schurer N.Y. Monger D.J. Hincenbergs M. Williams M.L. Fatty acid metabolism in human keratinocytes cultivated at an air–medium interface.J Invest Dermatol. 1989; 92: 196-202Abstract Full Text PDF PubMed Google Scholar;Madison et al., 1990Madison K.C. Swartzendruber D.C. Wertz P.W. Downing D.T. Sphingolipid metabolism in organotypic mouse keratinocyte cultures.J Invest Dermatol. 1990; 95: 657-664Crossref PubMed Scopus (24) Google Scholar). The amount of ceramide in the stratum corneum is regulated by the balance among the ceramide-generating enzymes including serine-palmitoyltransferase (Holleran et al., 1991Holleran W.M. Mao-Qiang M. Gao W.N. Menon G.K. Elias P.M. Feingold K.R. Sphingolipids are required for mammalian epidermal barrier function, inhibition of sphingolipid synthesis delays barrier recovery after acute perturbation.J Clin Invest. 1991; 88: 1338-1345Crossref PubMed Scopus (204) Google Scholar), sphingomyelinase (SMase) (Menon et al., 1986Menon G.K. Grayson S. Elias P.M. Cytochemical and biochemical localization of lipase and sphingomyelinase activity in mammalian epidermis.J Invest Dermatol. 1986; 86: 591-597Crossref PubMed Scopus (97) Google Scholar;Yamamura and Tezuka, 1990Yamamura T. Tezuka T. Change in sphingomyelinase activity in human epidermis during aging.J Dermatol Sci. 1990; 1: 79-84Abstract Full Text PDF PubMed Scopus (26) Google Scholar), and β-glucocerebrosidase (Holleran et al., 1992Holleran W.M. Takagi Y. Imokawa G. Jackson S. Lee J.M. Elias P.M. β-Glucocerebrosidase activity in murine epidermis: characterization and localization in relation to differentiation.J Lipid Res. 1992; 33: 1201-1209Abstract Full Text PDF PubMed Google Scholar) and the degradative enzyme ceramidase (Yada et al., 1995Yada Y. Higuchi K. Imokawa G. Purification and biochemical characterization of membrane-bound epidermal ceramidases from guinea pig skin.J Biol Chem. 1995; 270: 12677-12684Crossref PubMed Scopus (63) Google Scholar). Cultured keratinocytes were reported to have a high level of serine palmitoyltransferase activity, which catalyzes the synthesis of the long-chain base precursor of sphingolipids (Holleran et al., 1990Holleran W.M. Williams M.L. Gao W.N. Elias P.M. Serine-palmitoyl transferase activity in cultured human keratinocytes.J Lipid Res. 1990; 9: 1655-1661Google Scholar). Moreover, the activity of ceramide glucosyltransferase, the enzyme responsible for glucosyl-ceramide synthesis in cultured human keratinocytes, has been recently studied and characterized (Sando et al., 1996Sando G.N. Howard E.J. Madison K.C. Induction of ceramide glucosyltransferase activity in cultured human keratinocytes.J Biol Chem. 1996; 271: 22044-22051Crossref PubMed Scopus (50) Google Scholar). Both sphingomyelin (SM) and SMase, which hydrolyzes SM into ceramides, are present in the epidermis and are originally contained in lamellar bodies (Bowser and Gray, 1978Bowser P.A. Gray G.M. Sphingomyelinase in pig and human epidermis.J Invest Dermatol. 1978; 70: 331-335Crossref PubMed Scopus (55) Google Scholar;Yardley and Summerly, 1981Yardley H.J. Summerly R. Lipid composition and metabolism in normal and diseased epidermis.Pharmacol Ther. 1981; 13: 357-383Crossref PubMed Scopus (198) Google Scholar). An altered SM metabolism in the skin of patients with atopic dermatitis, which has been attributed to a deficient function of SMase and a parallel abnormal expression of SM acylase, has been reported; this could explain the ceramide deficiency and the marked vulnerability of the atopic skin to irritants or allergens (Murata et al., 1996Murata Y. Ogata J. Higaki Y. et al.Abnormal expression of sphingomyelin acylase in atopic dermatitis: an etiologic factor for ceramide deficiency?.J Invest Dermatol. 1996; 106: 1242-1249Crossref PubMed Scopus (193) Google Scholar). Moreover, recent reports suggest that the skin of patients with atopic dermatitis is colonized by ceramidase-secreting bacteria (i.e., Pseudomonas aeruginosa and/or related strains) and correlate the deficiency of ceramide in the horny layer of epidermis and the associated impairment of the barrier permeability with the presence of this ceramide-degradative enzyme (Okino et al., 1998Okino N. Tani M. Imayama S. Ito M. Purification and characterization of a novel ceramidase from Pseudomonas aeruginosa.J Biol Chem. 1998; 273: 14368-14373Crossref PubMed Scopus (84) Google Scholar;Ohnishi et al., 1999Ohnishi Y. Okino N. Ito M. Imayama S. Ceramidase activity in bacterial skin flora as a possible cause of ceramide deficiency in atopic dermatitis.Clin. Diagn Lab Immun. 1999; 6: 101-104PubMed Google Scholar). Interestingly,Rawlings et al., 1996Rawlings A.V. Davies A. Carlomusto M. et al.Effect of lactic acid isomers on keratinocyte ceramide synthesis, stratum corneum lipid levels and stratum corneum barrier function.Arch Dermatol Res. 1996; 288: 383-390Crossref PubMed Scopus (110) Google Scholar have reported the induction of a ceramide level enhancement either in keratinocytes in vitro and in skin in vivo, which was associated with an increased stratum corneum barrier and an increased effect in resisting skin xerosis. The authors hypothesized that lactic acid could be metabolized to acetate which in turn is used for ceramide biosynthesis in keratinocytes. Taken together, these findings strongly indicate that skin treatment with exogenous factors capable of increasing the levels of stratum corneum lipids, mainly ceramides, may improve barrier function and stratum corneum flexibility; consequently this may slow down, in healthy subjects, the skin aging process and offer advantages for patients with skin conditions due to a defective synthesis of lipids. As many bacteria possess high levels of SMase in their membrane, they may be particularly useful in inducing an increase of skin ceramide generation through SM hydrolysis. SM represents one of the most important lipid components of the mammalian plasma membrane and is preferentially localized in the external outleaf thus being readily available for its hydrolysis through an exogenous source of SMase, such as bacterial SMase. Indeed, bacterial SMase often has been used for the depletion of SM in a variety of cellular systems leading to the release of ceramide (Bettaieb et al., 1996Bettaieb A. Record M. Come M.G. Bras A.C. Chap H. Laurent G. Jaffrezouu J.P. Opposite effects of tumor necrosis factor alpha on the sphingomyelin-ceramide pathway in two myeloid leukemia cell lines: role of transverse sphingomyelin distribution in the plasma membrane.Blood. 1996; 88: 1465-1472Crossref PubMed Google Scholar;Wright et al., 1996Wright S.C. Zheng H. Zhong J. Tumor cell resistance to apoptosis due to a defect in the activation of sphingomyelinase and the 24 kDa apoptotic protease.FASEB J. 1996; 10: 325-332Crossref PubMed Scopus (63) Google Scholar;Flamigni et al., 1997Flamigni F. Faenza I. Marminoli S. et al.Inhibition of the expression of ornithine decarboxylase and c-Myc by cell-permeant ceramide in difluoromethylornithine-resistant leukaemia cells.Biochem J. 1997; 324: 783-789Crossref PubMed Scopus (22) Google Scholar;Zhang et al., 1997Zhang P. Liu B. Jenkins G.M. Hannun Y.A. Obeid L.M. Expression of neutral sphingomyelinase identifies a distinct pool of sphingomyelin involved in apoptosis.J Biol Chem. 1997; 272: 9609-9612Crossref PubMed Scopus (155) Google Scholar). Lactic acid bacteria are a group of bacteria belonging to a diverse genera used to bring about milk fermentation, and composed chiefly of bacteria whose primary metabolic end-product of carbohydrate metabolism is lactic acid; this in turn preserves milk by providing the acidity necessary for a tart flavor and for changes in the structure of casein to achieve syneresis and desired functional characteristics (Sanders, 1992Sanders M.E. Dairy products.Encyclopedia Microbiol. 1992; 2: 1-22Google Scholar;Johnson and Steele, 1997Johnson M.E. Steele J.L. Fermented dairy products.in: Doyle M.P. Beuchat L.R. Montville T.J. Food Microbiology. Academic Press, London1997: 581-594Google Scholar). Streptococcus salivarium subspeciem thermophilus belongs to this group of bacteria and is used to bring about the fermentation of several dairy products, mainly cheeses and yogurt (Sanders, 1992Sanders M.E. Dairy products.Encyclopedia Microbiol. 1992; 2: 1-22Google Scholar;Johnson and Steele, 1997Johnson M.E. Steele J.L. Fermented dairy products.in: Doyle M.P. Beuchat L.R. Montville T.J. Food Microbiology. Academic Press, London1997: 581-594Google Scholar) and does not form a constituent of normal skin resident microflora (James and Roth, 1992James W.D. Roth R.R. Skin microbiology.Encyclopedia Microbiol. 1992; 4: 23-32Google Scholar). The aim of this work was to assess the possibility of increasing ceramide levels either in vitro on cultured keratinocytes or in vivo on stratum corneum by treatment with a sonicated preparation of Streptococcus salivarium subspeciem thermophilus. Our results indicate that S. thermophilus treatment led to a significant increase in ceramide levels both in vitro and in vivo. If we consider that ceramides can maintain skin integrity, it is possible that topical administration of S. thermophilus (as a source of exogenous SMase able to hydrolyze skin SM) may consequently generate ceramide and phosphorylcholine, possibly leading to an improvement in barrier function and maintenance of stratum corneum flexibility; all these events may help the stratum corneum resist xerosis. The spontaneously immortalized human keratinocyte cell line HaCat (Boukamp et al., 1988Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3276) Google Scholar) was a gift from Dr. Diana Boraschi (Dompè Research Center, L'Aquila, Italy). Cells were grown in plastic culture dishes (Nunc, Wiesbaden, Germany) containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units penicillin per ml, 50 μg streptomycin per ml, and 50 μg gentamycin per ml. Media and culture reagents were obtained from Gibco (Berlin, Germany), penicillin, streptomycin, and gentamycin from Boehringer (Mannheim, Germany). Confluent cells were subcultured every 3 d after detaching the cells with a 0.1% trypsin/0.02% ethylenediamine tetraacetic acid solution. For the duration of the experiments, HaCaT cells were maintained in serum-free keratinocyte basal medium. Streptococcus thermophilus (strain S244), cultivated in 10% skimmed milk sterilized at 110°C for 30 min with 0.1% yeast extract, was obtained from Centro Ricerche YOMO (Milan, Italy), in a pure lyophilized form (10 cfu per g). Stocks of 1.7 g lyophilized S. thermophilus were resuspended in 5 ml phosphate-buffered solution (PBS), sonicated (3 min and 50 s, alternating 10 s sonication and 10 s pause) with a Vibracell sonicator (Sonic and Materials, Danbury, CT). For the in vitro experiments, sonicated bacterium suspension was added to keratinocyte cultures at 50 mg per 5 × 106 cells per 10 ml (final concentration). For the topical applications, the sonicated bacteria (1.7 g per 5 ml) were mixed with 20 ml of a base cream (Avant Garde, Sigma Tau, Pomezia, Rome, Italy). A base cream containing purified neutral SMase (nSMase) from Becillus cereus (Sigma) (4 ng per 50 μl PBS per ml base cream) was also prepared. Where indicated, bacterial extracts were preincubated for 60 min with 5 mM glutathione (GSH, Calbiochem) before addition to the cream. The GSH-containing creams were prepared immediately before use. S. thermophilusTen milligrams of lyophilized S. thermophilus were resuspended in 500 μl HEPES buffer 20 mM pH 7.4 containing 10 mM MgCl2, 2 mM ethylenediamine tetraacetic acid, 5 mM dithiothreitol, 0.1 mM Na3VO4, 0.1 mM Na2MoO4, 30 mM p-nitrophenylphosphate, 10 mM β-glycerophosphate, 750 μM adenosine triphosphate (ATP), 1 mM phenylmethylsulfonyl fluoride, 10 μM leupeptin, 10 μM pepstatin (Sigma), and 0.2% Triton X-100 (for the assay of nSMase) or 500 μl of 0.2% Triton X-100 [for the assay of acidic SMase (aSMase)] and sonicated as described above. Protein concentration was determined through the Pierce Micro BCA assay kit, with bovine serum albumin standards. For the assay of nSMase, different amounts of bacteria were incubated for 2 h at 37°C in HEPES buffer 20 mM pH 7.4 containing 1 mM MgCl2, and [N-methyl-14C]sphingomyelin (SM) (0.28 μCi per ml, specific activity 47 mCi per mmol; Amersham, Bucks., U.K.). The reaction was initiated by the addition of 40 μl of labeled SM, previously dried, resuspended in the assay buffer containing 3% Triton X-100, and solubilized by short bursts of sonication and vortexed. For the assay of aSMase, different amounts of sonicated bacteria were incubated for 2 h at 37°C in a buffer (200 μl final volume) containing 250 mM sodium acetate, 1 mM ethylenediamine tetraacetic acid, pH 5.0, and 40 μl of [N-methyl-14C]SM. Where indicated, the bacterial extracts were preincubated for 60 min with 5 mM GSH to inhibit nSMase activity, as previously described (Liu and Hannun, 1997Liu B. Hannun Y.A. Inhibition of the neutral magnesium-dependent sphingomyelinase by glutathione.J Biol Chem. 1997; 272: 16281-16287Crossref PubMed Scopus (274) Google Scholar;Liu et al., 1998Liu B. Andrieu-Abadie N. Levade T. Zhang P. Obeid L.M. Hannun Y.A. Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-α-induced cell death.J Biol Chem. 1998; 273: 11313-11320Crossref PubMed Scopus (318) Google Scholar). The reaction was stopped by the addition of 250 μl chloroform/methanol (2/1, by vol). The phospholipids were extracted by the addition of 800 μl chloroform/methanol (2/1, by vol), and 250 μl of H2O. After centrifugation at 10 978 × g for 15 min at 4°C, the aqueous phase was extracted again twice more with 500 μl chloroform. The organic phase, obtained in the different extraction steps, were collected and washed once with 1 ml chloroform/methanol/water (3/48/47, by vol), to remove totally free radioactive phosphorylcholine. The aqueous phases were collected, transferred to scintillation vials, and routinely counted by liquid scintillation counting. The counts per min represented the choline phosphate generated from SM hydrolysis. Correspondingly, the organic phase was analyzed on thin-layer chromatography (TLC) plates by using chloroform/methanol/ammonia hydroxide [7 M]/water (85/15/0.5/0.5, by vol). The hydrolysis of SM was quantitated by autoradiography and liquid scintillation. Subconfluent keratinocytes were incubated in the presence or absence of sonicated S. thermophilus (50 mg per 5 × 106 cells per 10 ml) at different times (0.25–18 h). After incubation, the cells were washed twice with ice-cold PBS (pH 7.2), resuspended in Tris/HCl buffer 20 mM pH 7.4, and harvested using a cell lifter (Costar, Cambridge, MA). The number of cells collected after the culture was not influenced by the presence of sonicated bacteria. After freeze-drying, the cells were sonicated (1 min and 50 s, alternating 10 s sonication and 10 s pause) and the protein concentration was determined using the Micro BCA protein assay reagent kit (Pierce, Rockford, IL), with bovine serum albumin standards. No significant differences in protein content were observed among control and treated cells. For the lipid extraction, 400 μl of methanol, 500 μl of chloroform, and 200 μl of water were added. Samples were stirred for 2 min on a vortex-mixer and centrifuged at 10 978 × g for 10 min. The extraction and centrifugation steps were repeated twice. Lipids, previously dried under nitrogen, were then incubated with Escherichia coli diacylglycerol kinase (DAG kinase assay kit and 32P-ATPγ, specific activity 3 Ci per mmol, Amersham) according to the manufacturer's instructions and applied to silica gel TLC plates using a TLC applicator (Camag, Berlin, Germany). Ceramide phosphate was then resolved using CHCl3/CH3OH/CH3COOH (65/15/5, vol/vol/vol) as solvent. Authentic ceramides from bovine brain (ceramide type III, nonhydroxy fatty acid ceramides; and ceramide type IV, hydroxy fatty acid ceramides; Sigma) were identified by autoradiography at Rf = 0.25 and Rf = 0.11, respectively. Specific radioactivity of ceramide-1-phosphate was determined by scintillation counting of corresponding spots scraped off the gel. Quantitative results for ceramide production were obtained by comparing the experimental values with a linear curve of the ceramide standards and are expressed as picomoles of ceramide-1-phosphate per 100 μg protein. Subconfluent HaCaT cells were incubated with pulse medium (DMEM containing 0.5 μCi [N-methyl-14C] choline per ml; Amersham; 55 mCi per mol) for 48 h at 37°C. The cells were then washed twice and cultured (5 × 106 cells per 10 ml) for 18 h with fresh medium in the presence or absence of sonicated S. thermophilus (50 mg per 10 ml, final concentration). After incubation, the cells were washed twice with ice-cold PBS (pH 7.2), resuspended in Tris/HCl buffer 20 mM pH 7.4, and harvested as described above. After lipid extraction, the organic phases, obtained from different extraction steps were collected, washed once with 1 ml of the solvent system containing chloroform/methanol/water (3/48/47, by vol), dried under nitrogen, and finally resuspended in 110 μl chloroform. Then, 10 μl aliquots of the chloroform phase were taken for scintillation counting and 100 μl were applied to silica gel TLC plates. Lipids were separated using chloroform/methanol/acetic acid/water (100/60/20/5, by vol) as the solvent system. Unlabeled lyso-phosphatidylcholine, SM, and phosphatidylcholine (Sigma) were used as standards and visualized in iodine vapor (Rf = 0.1, 0.26, and 0.6, respectively). Radioactive spots were visualized by autoradiography, scraped from the plate and counted by liquid scintillation. Assay of ceramide synthase activity was performed as previously described (Bose et al., 1995Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Ceramide synthase mediates Daunorubicin-induced apoptosis: an alternative mechanism for generating death signals.Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (770) Google Scholar). After treatment with sonicated S. thermophilus suspension, keratinocytes, collected (as described above), were resuspended in 300 μl of homogenization buffer (25 mM HEPES pH 7.4, 5 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 50 mM NaF, 10 μg leupeptin per ml, and 10 μg soybean trypsin inhibitor per ml), disrupted by sonication, and lysates were centrifuged at 800 × g for 5 min. Protein concentrations in the postnuclear supernatants were determined through the Micro BCA protein assay reagent kit (Pierce), with bovine serum albumin as the standard. Seventy-five microgram proteins were incubated in a 1 ml reaction mixture containing 2 mM MgCl2, 20 mM HEPES (pH 7.4), 20 μM defatted bovine serum albumin (Sigma), 20 μM dihydrosphingosine, 70 μM unlabeled palmitoyl-coenzyme A, and 3.6 μM (0.2 μCi) [1-14C]palmitoyl-coenzyme A (55 mCi per mmol; Amersham). Dihydrosphingosine was dried under nitrogen from a stock solution in 100% ethanol and dissolved with sonication in the reaction mixture prior to addition of cell extracts. The reaction was started by addition of palmitoyl-coenzyme A, incubated at 37°C for 1 h, and then stopped by extraction of lipids using 2 ml of chloroform/methanol (1/2, by vol). The lower phase was removed, concentrated under nitrogen, and applied to a silica gel 60 TLC plate. Dihydroceramide was resolved from free radiolabeled fatty acid using a solvent system of chloroform/methanol/3.5 M ammonium hydroxide (85/15/1), identified by autoradiography based on comigration with ceramide standards (stained with iodine vapor), and quantitated by liquid scintillation counting. The amount of palmitoyl-CoA consumed did not exceed 5% of total. To analyze cholesterol levels, lipids were extracted (as described above) from keratinocytes
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