title: Ceramide transfer protein deficiency compromises organelle function and leads to senescence in primary cells.
abstract: Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum (ER) to the Golgi complex. Its deficiency in mouse leads to embryonic death at E11.5. CERT deficient embryos die from cardiac failure due to defective organogenesis, but not due to ceramide induced apoptotic or necrotic cell death. In the current study we examined the effect of CERT deficiency in a primary cell line, namely, mouse embryonic fibroblasts (MEFs). We show that in MEFs, unlike in mutant embryos, lack of CERT does not lead to increased ceramide but causes an accumulation of hexosylceramides. Nevertheless, the defects due to defective sphingolipid metabolism that ensue, when ceramide fails to be trafficked from ER to the Golgi complex, compromise the viability of the cell. Therefore, MEFs display an incipient ER stress. While we observe that ceramide trafficking from ER to the Golgi complex is compromised, the forward transport of VSVG-GFP protein is unhindered from ER to Golgi complex to the plasma membrane. However, retrograde trafficking of the plasma membrane-associated cholera toxin B to the Golgi complex is reduced. The dysregulated sphingolipid metabolism also leads to increased mitochondrial hexosylceramide. The mitochondrial functions are also compromised in mutant MEFs since they have reduced ATP levels, have increased reactive oxygen species, and show increased glutathione reductase activity. Live-cell imaging shows that the mutant mitochondria exhibit reduced fission and fusion events. The mitochondrial dysfunction leads to an increased mitophagy in the CERT mutant MEFs. The compromised organelle function compromise cell viability and results in premature senescence of these MEFs.
authors.affiliation: Laboratory of Cell and Developmental Signaling, National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Raghavendra Pralhada
authors.initials: RP
authors.lastname: Rao
authors.affiliation: Laboratory of Cell and Developmental Signaling, National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Luana
authors.initials: L
authors.lastname: Scheffer
authors.affiliation: Laboratory of Cell and Developmental Signaling, National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Sargur M
authors.initials: SM
authors.lastname: Srideshikan
authors.affiliation: Laboratory of Cell and Developmental Signaling, National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Velayoudame
authors.initials: V
authors.lastname: Parthibane
authors.affiliation: Laboratory of Cell and Developmental Signaling, National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Teresa
authors.initials: T
authors.lastname: Kosakowska-Cholody
authors.affiliation: Laboratory of Proteomics and Analytical Technologies, Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
authors.forename: M Athar
authors.initials: MA
authors.lastname: Masood
authors.affiliation: Electron Microscopy Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
authors.forename: Kunio
authors.initials: K
authors.lastname: Nagashima
authors.affiliation: Optical Microscopy and Analysis Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
authors.forename: Prabhakar
authors.initials: P
authors.lastname: Gudla
authors.affiliation: Optical Microscopy and Analysis Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
authors.forename: Stephen
authors.initials: S
authors.lastname: Lockett
authors.affiliation: Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
authors.forename: Usha
authors.initials: U
authors.lastname: Acharya
authors.affiliation: Laboratory of Cell and Developmental Signaling, National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Jairaj K
authors.initials: JK
authors.lastname: Acharya
mesh_terms: D000818:Animals; D001692:Biological Transport; D049109:Cell Proliferation; D002470:Cell Survival; D016922:Cellular Senescence / Q000235:genetics*; D002518:Ceramides / Q000378:metabolism*; D002772:Cholera Toxin / Q000378:metabolism; D004622:Embryo, Mammalian; D004721:Endoplasmic Reticulum / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress; D005260:Female; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression; D006056:Golgi Apparatus / Q000378:metabolism; D050356:Lipid Metabolism / Q000235:genetics; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism* / Q000473:pathology; D061251:Primary Cell Culture; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency* / Q000235:genetics
chemical_list: D002518:Ceramides; D002772:Cholera Toxin; C535066:Col4a3bp protein, mouse; D017346:Protein Serine-Threonine Kinases