Sphingolipid metabolites such as sphingosine-1-phosphate (S1P) and ceramide modulate apoptosis during development and in response to stress. In general, ceramide promotes apoptosis, whereas S1P stimulates cell proliferation and protects against apoptosis. S1P is irreversibly degraded by the enzyme S1P lyase (SPL). In this study, we show a crucial role for SPL in mediating cellular responses to stress. SPL expression in HEK293 cells potentiated apoptosis in response to stressful stimuli including DNA damage. This effect seemed to be independent of ceramide generation but required SPL enzymatic activity and the actions of p38 MAP kinase, p53, p53-inducible death domain protein (PIDD), and caspase-2 as shown by molecular and chemical inhibition of each of these targets. Further, SPL expression led to constitutive activation of p38. Endogenous SPL expression was induced by DNA damage in WT cells, whereas SPL knockdown diminished apoptotic responses. Importantly, SPL expression was significantly downregulated in human colon cancer tissues in comparison with normal adjacent tissues, as determined by quantitative real-time PCR (Q-PCR) and immunohistochemical analysis. Down-regulation of S1P phosphatases was also observed, suggesting that colon cancer cells manifest a block in S1P catabolism. In addition, SPL expression and activity were down-regulated in adenomatous lesions of the Min mouse model of intestinal tumorigenesis. Taken together, these results indicate that endogenous SPL may play a physiological role in stress-induced apoptosis and provide an example of altered SPL expression in a human tumor. Our findings suggest that genetic or epigenetic changes affecting intestinal S1P metabolism may correlate with and potentially contribute to carcinogenesis.intestinal tumorigenesis ͉ Min mouse ͉ sphingolipid ͉ etoposide S phingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite and the final common product of complex sphingolipid metabolism. S1P acts through its cognate G proteincoupled receptors to inhibit apoptosis, regulate lymphocyte trafficking and to promote DNA synthesis, cell proliferation, cell migration, and angiogenesis (1, 2). The SPHK1 gene, which encodes the major sphingosine kinase responsible for S1P synthesis, can act as an oncogene in model systems (3). Further, parenteral administration of S1P-specific antibodies markedly slows human cancer xenograft progression and angiogenesis (4). S1P signaling has been implicated in the development of the drug resistant phenotype in cancer cells (5). Together, these findings strongly support a role for S1P signaling in promoting tumorigenesis and cancer progression. Despite these observations, evidence of genetic changes in human cancer tissues that would directly implicate S1P signaling in these processes is lacking. S1P is irreversibly degraded by the pyridoxal 5Ј-phosphatedependent enzyme, S1P lyase (SPL). SPL is highly conserved throughout evolution, is required for maintenance of physiological levels of S1P and other sphingolipid intermediates an...
FTY720 is a novel immunomodulatory agent that inhibits lymphocyte trafficking and prevents allograft rejection. FTY720 is phosphorylated in vivo, and the phosphorylated drug acts as agonist for a family of G protein-coupled receptors that recognize sphingosine 1-phosphate. Evidence suggests that FTY720-phosphate-induced activation of S1P 1 is responsible for its mechanism of action. FTY720 was rationally designed by modification of myriocin, a naturally occurring sphingoid base analog that causes immunosuppression by interrupting sphingolipid metabolism. In this study, we examined interactions between FTY720, FTY720-phosphate, and sphingosine-1-phosphate lyase, the enzyme responsible for irreversible sphingosine 1-phosphate degradation. FTY720-phosphate was stable in the presence of active sphingosine-1-phosphate lyase, demonstrating that the lyase does not contribute to FTY720 catabolism. Conversely, FTY720 inhibited sphingosine-1-phosphate lyase activity in vitro. Treatment of mice with FTY720 inhibited tissue sphingosine-1-phosphate lyase activity within 12 h, whereas lyase gene and protein expression were not significantly affected. Tissue sphingosine 1-phosphate levels remained stable or increased throughout treatment. These studies raise the possibility that disruption of sphingosine 1-phosphate metabolism may account for some effects of FTY720 on immune function and that sphingosine-1-phosphate lyase may be a potential target for immunomodulatory therapy.FTY720 is a novel immunosuppressive agent that modulates lymphocyte trafficking and prevents allograft rejection (1, 2). FTY720 treatment stimulates lymphocyte homing to peripheral lymph nodes and Peyer's patches, resulting in prolonged survival of allograft tissues and both prevention and treatment of various autoimmune diseases in animal models (3). FTY720 induces numerous effects on the immune system including inhibited egress of both naïve and activated CD4 ϩ , CD8 ϩ , and B lymphocytes from peripheral lymphoid organs and thymus, cysteinyl leukotriene-dependent T cell chemotaxis to lymph nodes, peripheral blood lymphopenia, egress of lymphocytes from the spleen, displacement of B cells from the marginal zone of the spleen, decreased 1 integrin expression on marginal zone B cells, homing of hematopoietic progenitor cells to the bone marrow, and decreased vascular permeability (4 -12). Importantly, while FTY720 prevents the migration of lymphocytes to allogeneic graft tissue and other sites of inflammation, it does not diminish the activation, proliferation, or effector functions of B and T lymphocytes in response to antigen stimulation (13). Toxicities associated with FTY720 are limited and distinct from those of other immunosuppressive drugs, which makes FTY720 an ideal candidate for combination immunosuppressive transplantation regimens.FTY720 was rationally designed based on chemical modifications of myriocin, a naturally occurring sphingoid base analog that causes immunosuppression by interrupting sphingolipid metabolism (14). FTY720 is phosphoryl...
Amide-linked indole-3-acetic acid (IAA) conjugates constitute approximately 90% of the IAA pool in the dicot Arabidopsis, whereas ester-linked conjugates and free IAA account for approximately 10% and 1%, respectively when whole seedlings are measured. We show here that IAA-aspartate Asp, IAA-glutamate (Glu), and IAA-glucose (Glc) are present at low levels in Arabidopsis. Nine-day-old wild-type Arabidopsis seedlings yielded 17.4 Ϯ 4.6 ng g Ϫ1 fresh weight IAA-Asp and 3.5 Ϯ 1.6 ng g Ϫ1 fresh weight IAA-Glu, and IAA-Glc was present at 7 to 17 ng g Ϫ1 fresh weight in 12-d-old wild-type seedlings. Total IAA content in 9-d-old Arabidopsis seedlings was 1,200 Ϯ 178 ng g Ϫ1 fresh weight, so these three IAA conjugates together made up only 3% of the conjugate pool throughout the whole plant. We detected less than wild-type levels of IAA-Asp and IAA-Glu (7.8 Ϯ 0.4 ng g Ϫ1 fresh weight and 1.8 Ϯ 0.3 ng g Ϫ1 fresh weight, respectively) in an Arabidopsis mutant that accumulates conjugated IAA. Our results are consistent with IAA-Asp, IAA-Glu, and IAA-Glc being either minor, transient, or specifically localized IAA metabolites under normal growth conditions and bring into question the physiological relevance of IAA-Asp accumulation in response to high concentrations of exogenous IAA.
Auxin measurements in plants are critical to understanding both auxin signaling and metabolic homeostasis. The most abundant natural auxin is indole-3-acetic acid (IAA). This protocol is for the precise, high-throughput determination of free IAA in plant tissue by isotope dilution analysis using gas chromatography-mass spectrometry (GC-MS). The steps described are as follows: harvesting of plant material; amino and polymethylmethacrylate solid-phase purification followed by derivatization with diazomethane (either manual or robotic); GC-MS analysis; and data analysis. [¹³C₆]IAA is the standard used. The amount of tissue required is relatively small (25 mg of fresh weight) and one can process more than 500 samples per week using an automated system. To extract eight samples, this procedure takes ∼3 h, whether performed manually or robotically. For processing more than eight samples, robotic extraction becomes substantially more time efficient, saving at least 0.5 h per additional batch of eight samples.
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