Re-branding Ceramide-1-phosphate: Not Just a Ceramide Metabolite
Open Access
- Author:
- Hankins, Jody
- Graduate Program:
- Pharmacology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 18, 2012
- Committee Members:
- Mark Kester, Ph D, Dissertation Advisor/Co-Advisor
Richard Bernard Mailman, Committee Member
Leo Fitzpatrick, Ph D, Committee Member
Channe D Gowda, Committee Member
Girish Subramanian, Committee Member - Keywords:
- Ceramide-1-phosphate
ceramide
sphingolipid
annexin a2
p11
Toll-like receptor 4
LPS - Abstract:
- Ceramide-1-phosphate (C-1-P), a bioactive sphingolipid produced from ceramide, promotes cell survival, proliferation, and migration. Historically, C-1-P was thought to oppose ceramide function by countering the accumulation of pro-apoptotic ceramide. Compared to other sphingolipids, little is known about C-1-P, so it is not surprising that C-1-P has long been considered just a “metabolite of ceramide”. These experiments reveal that C-1-P should no longer be considered a sphingolipid of secondary importance. The overall aim is to broaden the C-1-P knowledge base, which should guide rational predictions about acute and chronic effects of C-1-P modulation, a requirement for target validation for therapeutic intervention. To test the hypothesis that a cell-surface receptor mediates invasion of micro- and macrovascular endothelial cells through an extracellular matrix barrier, a protein/lipid binding screen was conducted to identify proteins that interact specifically with C-1-P. Described herein are the identification and characterization of a C-1-P interaction with the heterotetrameric protein complex of annexin a2/p11 in human primary vascular endothelial cells. A chemotaxis assay demonstrated that exogenously delivered C-1-P selectively mediates invasion of micro- and macrovascular endothelial cells through a Matrigel™ extracellular matrix barrier. In support of an interaction with a cell-surface protein, C-1-P-mediated invasion was independent of prostanoid production or enhanced cellular proliferation, processes known to be stimulated by intracellular accumulation of C-1-P. Demonstrating that C-1-P is readily secreted from endothelial cells, two-fold more C-1-P was detected by mass spectrometry in conditioned medium compared to cell lysates. C-1-P selectively interacted with the purified annexin a2/p11 heterotetramer using a lipid binding ELISA methodology, indicating an interaction. Additionally, immunoprecipitation of the p11 protein after exogenous C-1-P treatment demonstrated enrichment of associated C-1-P mass. Finally, siRNA-mediated knockdown of either annexin a2 or p11 protein inhibited C-1-P-mediated invasion, suggesting that annexin a2/p11 hetetrotetramer expression is required for this action of C-1-P. Identification of the annexin a2/p11 protein as a C-1-P-interacting protein represents the first report of a named putative C-1-P cell-surface receptor, substantiating C-1-P as a cellular signal independent of ceramide. In another related project, highlighting signaling induced by C-1-P, data suggested that C-1-P may also act upon another cell surface protein. Experiments were designed to test the hypothesis that C-1-P, acting independently of ceramide, modulates innate immune signaling via Toll-like receptor 4 (TLR4). C-1-P, long considered to promote inflammatory signaling, instead abrogated activation of TLR4 signaling by lipopolysaccharide (LPS). Specifically, C-1-P, but not ceramide, blocked LPS-stimulation at the level of: activation of the transcription factor NF-κB, multiple MAP kinase pathways, and release of pro-inflammatory cytokines. This action of C-1-P was specific to TLR4 signaling, since C-1-P exerted no effect on the same signaling pathways after stimulation with TNFα. Importantly, these results highlight C-1-P production, rather than ceramide reduction, as the causative agent. Contrary to the dogma that C-1-P is a “pro-inflammatory” lipid, these experiments revealed “anti-inflammatory” properties of exogenous C-1-P. Overall, such results suggest that these seemingly opposing viewpoints may converge on a new hypothesis indicating complex immunomodulation by C-1-P. In sum, C-1-P merits “re-branding” as a bioactive sphingolipid capable of transducing signals through cell-surface “receptors”, independent of ceramide.