Keratinocytes rapidly readjust ceramide-sphingomyelin homeostasis and contain a phosphatidylcholine-sphingomyelin transacylase.

Ceramide as central second messenger of the apoptosis-related sphingomyelin signaling pathway is a potential target for the control of cancer. A complex metabolizing network defines cell type and stage-specific final ceramide concentrations. Successful therapeutic control of ceramide levels requires a knowledge of multiple related turnover rates. The metabolism of ceramide and sphingomyelin was studied in keratinocytes under the condition of an unstimulated sphingomyelin signaling pathway. Preparations enriched in plasma membranes contain a neutral Mg(2+)-dependent sphingomyelinase and a Mg(2+)-independent sphingomyelin synthase that vigorously preserve balanced ceramide and sphingomyelin levels. Ceramide regulates neutral sphingomyelinase. Inhibition of sphingomyelin synthase by D609 treatment results in temporary loss of intercelluar contacts and in cellular shrinking. It is ineffective for sustained elevation of ceramide levels. Ceramide phosphorylating and deacylating activities are insignificant. Recently, fatty-acid remodeling in sphingomyelin was reported as likely to counteract the membrane-rigidifying effects of cholesterol. Keratinocytes transfer fluorescence labeled acyl-chains between phosphatidylcholine and sphingomyelin. A transferase of that kind would allow rapid adjustment of local lipid composition in response to acutely changed conditions. In addition, this transferase might have a function in the formation of the epidermal permeability barrier.

Human transaldolase-associated repetitive elements are transcribed by RNA polymerase III.

Repetitive elements flanked by exons 2 and 3 of the human transaldolase gene, thus termed transaldolase-associated repetitive elements, TARE, were identified in human DNA. Nonpolyadenylated TARE transcripts were detected by Northern blot analysis and cloned by reverse transcriptase-mediated polymerase chain reaction from human T lymphocytes. A dominant 1085-nucleotide long transcript, TARE-6, contained two adjacent Alu elements, a right monomer and a complete dimer, oriented opposite to the direction of transcription of the transaldolase gene. Reverse transcriptase-polymerase chain reaction and in vitro transcription analyses showed that transcription of TARE-6 proceeded in the orientation of the RNA pol III promoter of the Alu dimer and opposite to the orientation of the TAL-H gene. TAREs lacking RNA polymerase III promoter showed no transcriptional activity. In vitro transcription of TARE-6 was resistant to 1 microg/ml alpha-amanitin but sensitive to 100 microg/ml alpha-amanitin and tagetitoxin, suggesting involvement of RNA polymerase III. TAREs in both the transaldolase and HSAG-1 genomic loci were surrounded by TA target site duplications. Homologies between transaldolase and HSAG-1 break off internally at splice donor and acceptor sites. The results suggest RNA polymerase III-mediated transcription of TARE may be a source of repetitive elements, contributing to distinct genes and thus shaping the human genome.