Impaired TCR signaling through dysfunction of lipid rafts in sphingomyelin synthase 1 (SMS1)-knockdown T cells

Abstract

During T cell activation, TCRs cluster at the center of the T cell-antigen-presenting cell interface forming the central supramolecular activation cluster. Although it has been suggested that sphingolipid- and cholesterol-rich microdomains, termed lipid rafts, form platforms for the regulation and transduction of TCR signals, an actual role for membrane sphingomyelin (SM), a key component of lipid rafts, has not been reported. After cloning a gene responsible for SM synthesis, sphingomyelin synthase (SMS) 1, we established a SM-knockdown cell line (Jurkat-SMS1/kd) by transfection of SMS1-short-interfering RNA into Jurkat T cells, which is deficient in membrane expression of SM. Upon CD3 stimulation, expression of CD69 (the earliest leukocyte activation antigen), activation-induced cell adhesion and proliferation as well as TCR clustering was severely impaired in Jurkat-SMS1/kd cells. CD3-induced tyrosine phosphorylation and association of linker for activation of T cell with ZAP-70 and Grb2 and phosphorylation of protein kinase C (PKC) were also severely impaired in Jurkat-SMS1/kd cells. Finally, translocation of TCR, ZAP-70 and PKC into lipid rafts was markedly decreased in Jurkat-SMS1/kd cells. These findings indicate that membrane SM is crucial for TCR signal transduction, leading to full T cell activation through lipid raft function.

Caspase-mediated inhibition of sphingomyelin synthesis is involved in FasL-triggered cell death

“…Cycle threshold (C t ) values for SMS1 and SMS2 were 24.6±0.3 and 29.7±0.7 (mean ± S.E.M., n ¼ 3), respectively, suggesting that SMS1 is the predominant isoform in Jurkat cells, in agreement with a recent study. 33 Similar findings were reported in S49, a murine lymphoma cell line. 27 Considering the ability of FasL Sphingomyelin synthase 1 in FasL-induced apoptosis E Lafont et al to trigger a caspase-dependent inhibition of SMS activity in Jurkat cells, we hypothesized that SMS1 is a putative target of caspases.…”

“…Jurkat cell lines stably expressing siRNA against SMS1 have been recently characterized. 33 Expression of SMS1 mRNA in the SMS1 knockdown (KD) Jurkat variant (SMS1 KD) was reduced by approximately 40% as evaluated by quantitative real-time PCR 33 (Figure 6g). The role of SMS2 in FasL-induced apoptosis has also been investigated in HeLa cells.…”

“…32 Jurkat cells were stably transduced by a retroviral vector encoding either a scrambled (ATTGAAAAAGACACGCGCC) (control cells) or human SMS1 siRNA (GCCCAACTGCGAAGAATAA) (SMS1 KD cells). 33 HeLa cells stably overexpressing human SMS1 harboring the V5 tag at the C terminus 18 were kindly provided by Dr. J Holthuis (Utrecht, the Netherlands). The cells were transiently transfected with either 50 nM siRNA (siRNA On-Target SMARTpool) against SMS1 (Perbio, Brebières, France) or control (Non-Targeting SMARTpool) siRNA using Hiperfect reagent (Qiagen, Courtaboeuf, France), following the manufacturer's instructions.…”

Tales and Mysteries of the Enigmatic Sphingomyelin Synthase Family

“…78 SMS1 has been also implicated in raft-dependent activation of T-cell receptor in Jurkat cells. 79 By using Jurkat cells stably expressing shRNA targeting human SMS1, the authors established a cell line with impaired SM synthesis and levels in the plasma membrane. Stimulation of T-cell activation with CD3 revealed impaired expression of CD69 (early marker of leukocyte activation), adhesion, proliferation and TCR clustering and translocation to lipid rafts.…”

Loss of the Ceramide Transfer Protein Augments EGF Receptor Signaling in Breast Cancer

“…These membrane microdomains are thought to sequester certain membrane-associated signaling proteins while excluding others, thereby adding a spatial layer of control to signaling events (4,5). For example, in T lymphocytes lacking sphingomyelin (SM) synthase 1 (SMS1), Fas/CD95, and T-cell receptor clustering and subsequent activation of downstream signaling were shown to be compromised, suggesting a requirement for lipid rafts in the assembly of signaling complexes downstream of these receptors (6,7). Both ErbB1 and ErbB2/HER2 were found to be associated with lipid rafts in biochemical studies (8).…”