Possible roles of long-chain sphingomyelines and sphingomyelin synthase 2 in mouse macrophage inflammatory response.

To evaluate the precise role of sphingomyelin synthase 2 (SMS2) in sphingomyelin (SM) metabolism and their anti-inflammatory properties, we analyzed species of major SM and ceramide (Cer) (18:1, 18:0 sphingoid backbone, C14 - C26 N-acyl part) in SMS2 knockout and wild-type mouse plasma and liver using HPLC-MS. SMS2 deficiency significantly decreased very long chain SM (SM (d18:1/22:0) and SM (d18:1/24:0 or d18:0/24:1)) and increased very long chain Cer (Cer (d18:1/24:0 or d18:0/24:1) and Cer (d18:1/24:1)), but not long chain SM (SM (d18:1/16:0), SM (d18:1/18:0 or d18:0/18:1) and SM (d18:1/18:1)) in plasma. To examine the effects of SM on inflammation, we studied the role of very long chain SM in macrophage activation. Addition of SM (d18:1/24:0) strongly upregulated several macrophage activation markers, SM (d18:1/6:0) and Cer (d18:1/24:0) however, did not. It was suggested that very long chain SM but not long chain SM were decreased in SMS2-deficient mice liver and plasma. And the exogenously added very long chain SM (d18:1/24:0) could activate macrophages directly, suggesting a novel role of plasma very long chain SM in modulating macrophage activation and resulting inflammation.

Dynamic modification of sphingomyelin in lipid microdomains controls development of obesity, fatty liver, and type 2 diabetes.

Lipid microdomains or caveolae, small invaginations of plasma membrane, have emerged as important elements for lipid uptake and glucose homeostasis. Sphingomyelin (SM) is one of the major phospholipids of the lipid microdomains. In this study, we investigated the physiological function of sphingomyelin synthase 2 (SMS2) using SMS2 knock-out mice, and we found that SMS2 deficiency prevents high fat diet-induced obesity and insulin resistance. Interestingly, in the liver of SMS2 knock-out mice, large and mature lipid droplets were scarcely observed. Treatment with siRNA for SMS2 also decreased the large lipid droplets in HepG2 cells. Additionally, the siRNA of SMS2 decreased the accumulation of triglyceride in liver of leptin-deficient (ob/ob) mice, strongly suggesting that SMS2 is involved in lipid droplet formation. Furthermore, we found that SMS2 exists in lipid microdomains and partially associates with the fatty acid transporter CD36/FAT and with caveolin 1, a scaffolding protein of caveolae. Because CD36/FAT and caveolin 1 exist in lipid microdomains and are coordinately involved in lipid droplet formation, SMS2 is implicated in the modulation of the SM in lipid microdomains, resulting in the regulation of CD36/FAT and caveolae. Here, we established new cell lines, in which we can completely distinguish SMS2 activity from SMS1 activity, and we demonstrated that SMS2 could convert ceramide produced in the outer leaflet of the plasma membrane into SM. Our findings demonstrate the novel and dynamic regulation of lipid microdomains via conformational changes in lipids on the plasma membrane by SMS2, which is responsible for obesity and type 2 diabetes.

Effect of the content of unmethylated CpG dinucleotides in plasmid DNA on the sustainability of transgene expression.

BACKGROUND: Nonviral gene transfer generally suffers from short-term expression of transgenes. We have previously demonstrated that plasmids with reduced CpG content exhibited a more prolonged expression of murine interferon (IFN)-beta or IFN-gamma, which was effective in inhibiting metastatic tumor growth. A further extension of the duration of transgene expression could be achieved by controlling the number and location of CpG motifs in plasmid DNA. METHODS: Luciferase-expressing plasmids with differing CpG content were injected into the tail vein of mice by the hydrodynamic injection method. The effects of CpG content on the duration of transgene expression were examined, focusing on cytosine methylation and pro-inflammatory cytokines. Based on the findings, IFN-gamma-expressing plasmids were constructed and their transgene expression and inhibitory effect on pulmonary metastasis were evaluated. RESULTS: Plasmids with a few CpG motifs showed a prolonged luciferase activity in the liver. Methylation of CpG motifs in plasmids reduced the expression and the extent of this reduction was greater for plasmids with a high CpG content. Pro-inflammatory cytokines hardly affected the expression. pCpG-Mu gamma, the IFN-gamma-expressing plasmid, which contains 20 CpG motifs only in the cDNA region, exhibited a sustained IFN-gamma concentration at therapeutic levels, and had a great inhibitory effect on the pulmonary metastasis of tumor cells. CONCLUSIONS: The duration of transgene expression of IFN-gamma was successfully increased by reducing the CpG content of IFN-expressing plasmid vector, which resulted in an increased anticancer activity of IFN gene transfer.

Improved anti-cancer effect of interferon gene transfer by sustained expression using CpG-reduced plasmid DNA.

Plasmid DNA (pDNA) expressing mouse interferon (IFN)-beta or IFN-gamma (pCMV-Mu beta and pCMV-Mu gamma, respectively) has been shown to be effective in inhibiting the growth of colon carcinoma CT-26 cells in the liver (Kobayashi et al., Molecular Therapy 2002;6:737-44). The therapeutic effect of such IFN gene transfer could be significantly increased by the sustained expression of IFNs. In the present study, CpG-reduced pDNA encoding IFN-beta or IFN-gamma (pGZB-Mu beta and pGZB-Mu gamma, respectively) was constructed. pCMV-Mu beta and pCMV-Mu gamma were used as conventional CpG-replete pDNAs. Each pDNA was injected into the tail vein of mice by the hydrodynamics-based procedure. An injection of pGZB-Mu beta resulted in very high IFN-beta activities in the serum for at least 24 hr after injection, whereas the IFN-beta activity after pCMV-Mu beta injection declined quickly. About a 14-fold greater amount of IFN-beta was produced from pGZB-Mu beta than from pCMV-Mu beta. pGZB-Mu beta markedly inhibited the pulmonary metastasis of CT-26 cells. Similar, but more marked results were obtained with pGZB-Mu gamma: it increased the area under the concentration-time curve by more than a 60-fold and the mean residence time of IFN-gamma 4-fold compared with pCMV-Mu gamma. The survival time of the pGZB-Mu gamma-treated mice was significantly (p<0.05) longer than that of the saline- or pCMV-Mu gamma-treated mice. These results indicate that long-term expression of IFN can be achieved by CpG-reduced pDNA and sustained IFN gene expression results in enhanced therapeutic effects of IFN gene transfer against tumor metastasis.