The epidermal permeability barrier of premature infants matures rapidly following birth. Previous studies suggest that air exposure could contribute to this acceleration, because: (i) development of a structurally and functionally mature barrier accelerates when fetal rat skin explants are incubated at an air-medium interface, and (ii) occlusion with a water-impermeable membrane prevents this acceleration. To investigate further the effects of air exposure on epidermal barrier ontogenesis, we compared the activities of several key enzymes of lipid metabolism and gene expression of protein markers of epidermal differentiation in fetal rat skin explants grown immersed versus air exposed. The rate-limiting enzymes of cholesterol (HMG CoA reductase) and ceramide (serine palmitoyl transferase) synthesis were not affected. In contrast, the normal developmental increases in activities of glucosylceramide synthase and cholesterol sulfotransferase, responsible for the synthesis of glucosylceramides and cholesterol sulfate, respectively, were accelerated further by air exposure. Additionally, two enzymes required for the final stages of barrier maturation and essential for normal stratum corneum function, beta-glucocerebrosidase, which converts glucosylceramide to ceramide, and steroid sulfatase, which desulfates cholesterol sulfate, also increased with air exposure. Furthermore, filaggrin and loricrin mRNA levels, and filaggrin, loricrin, and involucrin protein levels all increased with air exposure. Finally, occlusion with a water-impermeable membrane prevented both the air-exposure-induced increase in lipid enzyme activity, and the expression of loricrin, filaggrin, and involucrin. Thus, air exposure stimulates selected lipid metabolic enzymes and the gene expression of key structural proteins in fetal epidermis, providing a biochemical basis for air-induced acceleration of permeability barrier maturation in premature infants. (+info)
(2/204) The identification of myriocin-binding proteins.
BACKGROUND: Myriocin is a natural product that potently induces apoptosis of a murine cytotoxic T lymphocyte cell line (CTLL-2) and inhibits a serine palmitoyltransferase (SPT) activity that has been detected in cell extracts and is thought to initiate sphingolipid biosynthesis. Because SPT has never been biochemically purified and a comprehensive appraisal of myriocin-binding proteins has not been conducted, we isolated specific targets using myriocin-based affinity chromatography. RESULTS: Myriocin derivatives were synthesized and evaluated using CTLL-2 proliferation and SPT activity assays. Guided by these results, affinity chromatography matrices were prepared and two specific myriocin-binding proteins were isolated from CTLL-2 lysates. Analyses of these polypeptides establish conclusively that they are murine LCB1 and LCB2, mammalian homologs of two yeast proteins that have been genetically linked to sphingolipid biosynthesis. CONCLUSION: The ability of myriocin-containing matrices to bind factors that have SPT activity and the exclusive isolation of LCB1 and LCB2 as myriocin-binding proteins demonstrates that the two proteins are directly responsible for SPT activity and that myriocin acts directly upon these polypeptides. (+info)
(3/204) Activation of the de novo biosynthesis of sphingolipids mediates angiotensin II type 2 receptor-induced apoptosis.
This study examines the role of sphingolipids in mediating the apoptosis of PC12W cells induced by the angiotensin II type 2 (AT2) receptor. PC12W cells express abundant AT2 receptor but not angiotensin II type 1 receptor and undergo apoptosis when stimulated by angiotensin II. AT2 receptor-induced ceramide accumulation preceded the onset of caspase 3 activation and DNA fragmentation. AT2 receptor-induced ceramide accumulation did not result from the degradation of complex sphingolipids (SL) such as sphingomyelin or glycosphingolipids, as no changes in neutral or acidic sphingomyelinase activities, sphingomyelin level, nor in cellular glycolipid composition were observed. AT2 receptor activated serine palmitoyltransferase with a maximum time of 24 h after angiotensin II stimulation. The AT2 receptor-induced accumulation of ceramide was blocked by inhibitors of the de novo pathway of SL synthesis, beta-chloro-L-alanine and fumonisin B1. Inhibition of the de novo biosynthesis of SLs by fumonisin B1 and beta-chloro-L-alanine completely abrogated the AT2 receptor-mediated apoptosis. Pertussis toxin and orthovanadate blocked AT2 receptor-mediated ceramide production. Taken together our data demonstrate that in PC12W cells the stimulation of AT2 receptor induces the activation of de novo pathway, and a metabolite of this pathway, possibly ceramide, mediates AT2 receptor-induced apoptosis. (+info)
(4/204) De novo synthesis of sphingolipids is required for cell survival by down-regulating c-Jun N-terminal kinase in Drosophila imaginal discs.
Mitogen-activated protein kinase (MAPK) is a conserved eukaryotic signaling factor that mediates various signals, cumulating in the activation of transcription factors. Extracellular signal-regulated kinase (ERK), a MAPK, is activated through phosphorylation by the kinase MAPK/ERK kinase (MEK). To elucidate the extent of the involvement of ERK in various aspects of animal development, we searched for a Drosophila mutant which responds to elevated MEK activity and herein identified a lace mutant. Mutants with mild lace alleles grow to become adults with multiple aberrant morphologies in the appendages, compound eye, and bristles. These aberrations were suppressed by elevated MEK activity. Structural and transgenic analyses of the lace cDNA have revealed that the lace gene product is a membrane protein similar to the yeast protein LCB2, a subunit of serine palmitoyltransferase (SPT), which catalyzes the first step of sphingolipid biosynthesis. In fact, SPT activity in the fly expressing epitope-tagged Lace was absorbed by epitope-specific antibody. The number of dead cells in various imaginal discs of a lace hypomorph was considerably increased, thereby ectopically activating c-Jun N-terminal kinase (JNK), another MAPK. These results account for the adult phenotypes of the lace mutant and suppression of the phenotypes by elevated MEK activity: we hypothesize that mutation of lace causes decreased de novo synthesis of sphingolipid metabolites, some of which are signaling molecules, and one or more of these changes activates JNK to elicit apoptosis. The ERK pathway may be antagonistic to the JNK pathway in the control of cell survival. (+info)
(5/204) Tsc3p is an 80-amino acid protein associated with serine palmitoyltransferase and required for optimal enzyme activity.
Serine palmitoyltransferase catalyzes the first step of sphingolipid synthesis, condensation of serine and palmitoyl CoA to form the long chain base 3-ketosphinganine. The LCB1/TSC2 and LCB2/TSC1 genes encode homologous proteins of the alpha-oxoamine synthase family required for serine palmitoyltransferase activity. The other alpha-oxoamine synthases are soluble homodimers, but serine palmitoyltransferase is a membrane-associated enzyme composed of at least two subunits, Lcb1p and Lcb2p. Here, we report the characterization of a third gene, TSC3, required for optimal 3-ketosphinganine synthesis in Saccharomyces cerevisiae. S. cerevisiae cells lacking the TSC3 gene have a temperature-sensitive lethal phenotype that is reversed by supplying 3-ketosphinganine, dihydrosphingosine, or phytosphingosine in the growth medium. The tsc3 mutant cells have severely reduced serine palmitoyltransferase activity. The TSC3 gene encodes a novel 80-amino acid protein with a predominantly hydrophilic amino-terminal half and a hydrophobic carboxyl terminus that is membrane-associated. Tsc3p coimmunoprecipitates with Lcb1p and/or Lcb2p but does not bind as tightly as Lcb1p and Lcb2p bind to each other. Lcb1p and Lcb2p remain tightly associated with each other and localize to the membrane in cells lacking Tsc3p. However, Lcb2p is unstable in cells lacking Lcb1p and vice versa. (+info)
(6/204) Purification of the serine palmitoyltransferase complex responsible for sphingoid base synthesis by using affinity peptide chromatography techniques.
Serine palmitoyltransferase (SPT), a membrane-bound enzyme of the endoplasmic reticulum, catalyzes the condensation of palmitoyl coenzyme A (CoA) and L-serine to produce 3-ketodihydrosphingosine. This enzyme contains at least two different subunits, named the LCB1 and LCB2 proteins. In the present study, we expressed a FLAG- and His(6) peptide-tagged version of the hamster LCB1 protein in a Chinese hamster ovary cell mutant strain lacking the endogenous LCB1 subunit and purified SPT from the cells near to homogeneity by affinity peptide chromatography. The endogenous LCB2 protein was co-purified with the tagged LCB1 protein in purification of SPT. In various aspects, including optimum pH, acyl-CoA specificity, and sphingofungin sensitivity, the activity of purified SPT was consistent with the activity detected in lysates of wild-type Chinese hamster ovary cells. The optimum concentration of palmitoyl-CoA for 3-ketodihydrosphingosine formation by purified SPT was approximately 25 microM, and the apparent K(m) of L-serine was 0.28 mM. Competition analysis of the SPT reaction with various serine analogs showed that all of the amino, carboxyl, and hydroxyl groups of L-serine were responsible for the substrate recognition of the enzyme. SDS-polyacrylamide gel electrophoretic analysis of purified SPT, together with immunoprecipitation analysis of metabolically labeled LCB proteins, strongly suggested that the SPT enzyme consisted of the LCB1 and LCB2 proteins with a stoichiometry of 1:1. (+info)
(7/204) Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis.
The de novo pathway of sphingolipid synthesis has been identified recently as a novel means of generating ceramide during apoptosis. Furthermore, it has been suggested that the activation of dihydroceramide synthase is responsible for increased ceramide production through this pathway. In this study, accumulation of ceramide mass in Molt-4 human leukemia cells by the chemotherapy agent etoposide was found to occur primarily due to activation of the de novo pathway. However, when the cells were labeled with a substrate for dihydroceramide synthase in the presence of etoposide, there was no corresponding increase in labeled ceramide. Further investigation using a labeled substrate for serine palmitoyltransferase, the rate-limiting enzyme in the pathway, resulted in an accumulation of label in ceramide upon etoposide treatment. This result suggests that the activation of serine palmitoyltransferase is the event responsible for increased ceramide generation during de novo synthesis initiated by etoposide. Importantly, the ceramide generated from de novo synthesis appears to have a distinct function from that induced by sphingomyelinase action in that it is not involved in caspase-induced poly (ADP-ribose)polymerase proteolysis but does play a role in disrupting membrane integrity in this model system. These results implicate serine palmitoyltransferase as the enzyme controlling de novo ceramide synthesis during apoptosis and begin to define a unique function of ceramide generated from this pathway. (+info)
(8/204) Peroxisome proliferator-activated receptor-alpha enhances lipid metabolism in a skin equivalent model.
Peroxisome proliferator-activated receptors are involved in certain cell types such as adipocytes and hepatocytes, in the control of several pathways of lipid synthesis or catabolism by regulating the gene expression level of key lipid metabolizing enzymes. As the epidermis exhibits an extensive lipid metabolism necessary for the establishment of the barrier function, we have examined the role of peroxisome proliferator-activated receptor-alpha activation in this process. Living skin equivalents were treated with Wy 14,643, a selective peroxisome proliferator- activated receptor-alpha ligand, which enhanced greatly the synthesis of membrane coating granules, the organelles specialized in the processing of stratum corneum lipids. Also, the overall stratum corneum neutral lipid content assessed by Oil red O staining was increased. A detailed analysis of the lipid species present in the reconstructed epidermis showed that peroxisome proliferator-activated receptor-alpha activation increased the synthesis of ceramides and cholesterol derivatives, thought to be essential structural components of the permeability barrier. A synergistic effect was observed on lipid synthesis when peroxisome proliferator-activated receptor-alpha and retinoid X receptor were simultaneously activated by selective ligands. Furthermore, activation of peroxisome proliferator-activated receptor-alpha led to increased mRNA expression of several key enzymes of ceramide and cholesterol metabolism. An increase of serine-palmitoyl transferase and of beta-glucocerebrosidase enzymatic activity was also demonstrated. Altogether, these results show that peroxisome proliferator-activated receptor-alpha is a key transcription factor involved in the control of the epidermal lipid barrier. (+info)