Autotaxin (ATX) is a secreted lysophospholipase D that generates the bioactive lipid mediator lysophosphatidic acid (LPA). We and others have reported that ATX binds to integrins, but the function of ...ATX-integrin interactions is unknown. The recently reported crystal structure of ATX suggests a role for the solvent-exposed surface of the N-terminal tandem somatomedin B-like domains in binding to platelet integrin αIIbβ3. The opposite face of the somatomedin B-like domain interacts with the catalytic phosphodiesterase (PDE) domain to form a hydrophobic channel through which lysophospholipid substrates enter and leave the active site. Based on this structure, we hypothesize that integrin-bound ATX can access cell surface substrates and deliver LPA to cell surface receptors. To test this hypothesis, we investigated the integrin selectivity and signaling pathways that promote ATX binding to platelets. We report that both platelet β1 and β3 integrins interact in an activation-dependent manner with ATX via the SMB2 domain. ATX increases thrombin-stimulated LPA production by washed platelets ∼10-fold. When incubated under conditions to promote integrin activation, ATX generates LPA from CHO cells primed with bee venom phospholipase A2, and ATX-mediated LPA production is enhanced more than 2-fold by CHO cell overexpression of integrin β3. The effects of ATX on platelet and cell-associated LPA production, but not hydrolysis of small molecule or detergent-solubilized substrates, are attenuated by point mutations in the SMB2 that impair integrin binding. Integrin binding therefore localizes ATX activity to the cell surface, providing a mechanism to generate LPA in the vicinity of its receptors.
The ORM1 (Saccharomyces cerevisiae)-like proteins (ORMDLs) and their yeast orthologs, the Orms, are negative homeostatic regulators of the initiating enzyme in sphingolipid biosynthesis, serine ...palmitoyltransferase (SPT). Genome-wide association studies have established a strong correlation between elevated expression of the endoplasmic reticulum protein ORMDL3 and risk for childhood asthma. Here we test the notion that elevated levels of ORMDL3 decrease sphingolipid biosynthesis. This was tested in cultured human bronchial epithelial cells (HBECs) (an immortalized, but untransformed, airway epithelial cell line) and in HeLa cells (a cervical adenocarcinoma cell line). Surprisingly, elevated ORMDL3 expression did not suppress de novo biosynthesis of sphingolipids. We determined that ORMDL is expressed in functional excess relative to SPT at normal levels of expression. ORMDLs and SPT form stable complexes that are not increased by elevated ORMDL3 expression. Although sphingolipid biosynthesis was not decreased by elevated ORMDL3 expression, the steady state mass levels of all major sphingolipids were marginally decreased by low level ORMDL3 over-expression in HBECs. These data indicate that the contribution of ORMDL3 to asthma risk may involve changes in sphingolipid metabolism, but that the connection is complex.
Sphingosine-1-phosphate (S1P) is an intracellularly generated bioactive lipid essential for development, vascular integrity, and immunity. These functions are mediated by S1P-selective cell surface ...G-protein coupled receptors. S1P signaling therefore requires extracellular release of this lipid. Several cell types release S1P and evidence for both plasma membrane transporter-mediated and vesicle-dependent secretion has been presented. Platelets are an important source of S1P and can release it in response to agonists generated at sites of vascular injury. S1P release from agonist-stimulated platelets was measured in the presence of a carrier molecule (albumin) using HPLC-MS/MS. The kinetics and agonist-dependence of S1P release were similar to that of other granule cargo e.g. platelet factor IV (PF4). Agonist-stimulated S1P release was defective in platelets from Unc13dJinx (Munc13-4 null) mice demonstrating a critical role for regulated membrane fusion in this process. Consistent with this observation, platelets efficiently converted fluorescent NBD-sphingosine to its phosphorylated derivative which accumulated in granules. Fractionation of platelet organelles revealed the presence of S1P in both the plasma membrane and in α-granules. Resting platelets contained a second pool of constitutively releasable S1P that was more rapidly labeled by exogenously added sphingosine. Our studies indicate that platelets contain two pools of S1P that are released extracellularly: a readily-exchangeable, metabolically active pool of S1P, perhaps in the plasma membrane, and a granular pool that requires platelet activation and regulated exocytosis for release.
•Sphingosine-1-phosphate (S1P) promotes endothelial integrity.•Platelets release S1P both constitutively and in an activation-dependent manner.•Platelets have non-granular and granular S1P pools.•Release of the granular pool of S1P involves regulated exocytosis.
Coplanar polychlorinated biphenyls (PCBs) promote adipocyte inflammation and impair glucose homeostasis in lean mice. The diabetes-promoting effects of lipophilic PCBs have been observed only during ...weight loss in obese mice. The molecular mechanisms linking PCB exposures to impaired glucose metabolism are unclear.
In this study we tested the hypothesis that coplanar PCBs act at adipocyte aryl hydrocarbon receptors (AhRs) to promote adipose inflammation and impair glucose homeostasis in lean mice and in obese mice during weight loss.
PCB-77 administration impaired glucose and insulin tolerance in LF (low fat diet)-fed control (AhR(fl/fl)) mice but not in adipocyte AhR-deficient mice (AhR(AdQ)). Unexpectedly, AhR(AdQ) mice exhibited increased fat mass when fed a standard LF or high fat (HF) diet. In mice fed a HF diet, both genotypes became obese, but AhR(AdQ) mice administered vehicle (VEH) exhibited increased body weight, adipose mass, adipose inflammation, and impaired glucose tolerance compared with AhR(fl/fl) controls. Impairment of glucose homeostasis in response to PCB-77 was not observed in obese mice of either genotype. However, upon weight loss, AhR(fl/fl) mice administered PCB-77 exhibited increased abundance of adipose tumor necrosis factor-α (TNF-α) mRNA and impaired glucose homeostasis compared with those administered VEH. In contrast, PCB-77 had no effect on TNF-α or glucose homeostasis in AhR(AdQ) mice exhibiting weight loss.
Our results demonstrate that adipocyte AhR mediates PCB-induced adipose inflammation and impairment of glucose homeostasis in mice. Moreover, deficiency of AhR in adipocytes augmented the development of obesity, indicating that endogenous ligand(s) for AhR regulate adipose homeostasis.
Fatty acid synthase (FASN) and ATP-citrate lyase, key enzymes of de novo lipogenesis, are significantly upregulated and activated in many cancers and portend poor prognosis. Even though the role of ...lipogenesis in providing proliferative and survival advantages to cancer cells has been described, the impact of aberrant activation of lipogenic enzymes on cancer progression remains unknown. In this study, we found that elevated expression of FASN is associated with advanced stages of colorectal cancer (CRC) and liver metastasis, suggesting that it may play a role in progression of CRC to metastatic disease. Targeted inhibition of lipogenic enzymes abolished expression of CD44, a transmembrane protein associated with metastases in several cancers including CRC. In addition, inhibition of lipogenic enzymes and reduced expression of CD44 attenuated the activation of MET, Akt, FAK, and paxillin, which are known to regulate adhesion, migration, and invasion. These changes were consistent with an observed decrease in migration and adhesion of CRC cells in functional assays and with reorganization of actin cytoskeleton upon FASN inhibition. Despite the modest effect of FASN inhibition on tumor growth in xenografts, attenuation of lipogenesis completely abolished establishment of hepatic metastasis and formation of secondary metastasis. Together, our findings suggest that targeting de novo lipogenesis may be a potential treatment strategy for advanced CRC.
Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, ...cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.
Plasma sphingosine-1-phosphate (S1P) regulates vascular permeability, and plasma and lymph S1P guide lymphocyte egress from lymphoid organs. S1P is made intracellularly, and little is known about how ...S1P is delivered into circulatory fluids. Here, we find that mice without the major facilitator superfamily transporter Spns2 have a profound reduction in lymph S1P, but only a minor decrease in plasma S1P. Spns2-deficient mice have a redistribution of lymphocytes from the spleen to lymph nodes and a loss of circulating lymphocytes, consistent with normal egress from the spleen directed by plasma S1P and blocked egress from lymph nodes directed by lymph S1P. Spns2 is needed in endothelial cells to supply lymph S1P and support lymphocyte circulation. As a differential requirement for lymph and blood S1P, Spns2 may be an attractive target for immune suppressive drugs.
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► The transporter Spns2 is required to supply lymph, but not plasma, S1P ► Spns2-deficient mice have disrupted peripheral lymphocyte circulation ► Spns2 is required in endothelial cells to secrete lymph S1P and support trafficking
Blood sphingosine-1-phosphate (S1P) maintains vascular stability, and blood and lymph S1P guide lymphocyte exit from lymphoid organs. Schwab and colleagues demonstrate that while endothelial cells require the transporter Spns2 to secrete S1P into lymph, red blood cells use a Spns2-independent mechanism for S1P transport into blood. As a differential requirement for lymph and blood S1P, Spns2 may be an attractive target for immune-suppressive drugs that inhibit lymphocyte egress while minimizing vascular side effects.
Autotaxin (ATX) is a secreted nucleotide pyrophosphatase/phosphodiesterase that functions as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA), a mitogen, ...chemoattractant, and survival factor for many cell types. The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation, fibrotic diseases and tumor progression, making this system an attractive target for therapy. However, potent and selective nonlipid inhibitors of ATX are currently not available. By screening a chemical library, we have identified thiazolidinediones that selectively inhibit ATX-mediated LPA production both in vitro and in vivo. Inhibitor potency was approximately 100-fold increased (IC₅₀ ~ 30 nM) after the incorporation of a boronic acid moiety, designed to target the active-site threonine (T210) in ATX. Intravenous injection of this inhibitor into mice resulted in a surprisingly rapid decrease in plasma LPA levels, indicating that turnover of LPA in the circulation is much more dynamic than previously appreciated. Thus, boronic acid-based small molecules hold promise as candidate drugs to target ATX.
Ceramide-1-phosphate (C1P) is a bioactive lipid that, in contrast to ceramide, is an antiapoptotic molecule released from cells that are damaged and "leaky." As reported recently, C1P promotes ...migration of hematopoietic cells. In this article, we tested the hypothesis that C1P released upon tissue damage may play an underappreciated role in chemoattraction of various types of stem cells and endothelial cells involved in tissue/organ regeneration. We show for the first time that C1P is upregulated in damaged tissues and chemoattracts bone marrow (BM)-derived multipotent stromal cells, endothelial progenitor cells, and very small embryonic-like stem cells. Furthermore, compared to other bioactive lipids, C1P more potently chemoattracted human umbilical vein endothelial cells and stimulated tube formation by these cells. C1P also promoted in vivo vascularization of Matrigel implants and stimulated secretion of stromal cell-derived factor-1 from BM-derived fibroblasts. Thus, our data demonstrate, for the first time, that C1P is a potent bioactive lipid released from damaged cells that potentially plays an important and novel role in recruitment of stem/progenitor cells to damaged organs and may promote their vascularization.
Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for ...sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS.
Background: Sphingolipids play an important role in glucose homeostasis.
Results: High glucose induces SphK activity, leading to increases in S1P levels and stimulation of insulin secretion.
Conclusion: SphK activity and S1P levels are critical for glucose-stimulated insulin secretion.
Significance: The presented data uncover a new role for SphK and S1P in regulation of insulin secretion by glucose.
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