Ceramide Metabolism Regulated by Sphingomyelin Synthase 2 Is Associated with Acquisition of Chemoresistance via Exosomes in Human Leukemia Cells.

Ceramide levels controlled by the sphingomyelin (SM) cycle have essential roles in cancer cell fate through the regulation of cell proliferation, death, metastasis, and drug resistance. Recent studies suggest that exosomes confer cancer malignancy. However, the relationship between ceramide metabolism and exosome-mediated cancer malignancy is unclear. In this study, we elucidated the role of ceramide metabolism via the SM cycle in exosomes and drug resistance in human leukemia HL-60 and adriamycin-resistant HL-60/ADR cells. HL-60/ADR cells showed significantly increased exosome production and release compared with parental chemosensitive HL-60 cells. In HL-60/ADR cells, increased SM synthase (SMS) activity reduced ceramide levels, although released exosomes exhibited a high ceramide ratio in both HL-60- and HL-60/ADR-derived exosomes. Overexpression of SMS2 but not SMS1 suppressed intracellular ceramide levels and accelerated exosome production and release in HL-60 cells. Notably, HL-60/ADR exosomes conferred cell proliferation and doxorubicin resistance properties to HL-60 cells. Finally, microRNA analysis in HL-60 and HL-60/ADR cells and exosomes showed that miR-484 elevation in HL-60/ADR cells and exosomes was associated with exosome-mediated cell proliferation. This suggests that intracellular ceramide metabolism by SMS2 regulates exosome production and release, leading to acquisition of drug resistance and enhanced cell proliferation in leukemia cells.

A novel mechanism of thrombocytopenia by PS exposure through TMEM16F in sphingomyelin synthase 1 deficiency.

Sphingomyelin synthase 1 (SMS1) contributes to the generation of membrane sphingomyelin (SM) and affects SM-mediated physiological functions. Here, we describe the hematologic phenotypes, such as reduced circulating platelets and dysfunctional hemostasis, in SMS1-deficient (SMS1-KO) mice. SMS1-KO mice display pathologic manifestations related to idiopathic thrombocytopenia (ITP), including relatively high amounts of peripheral blood reticulated platelets, enhanced megakaryopoiesis in the bone marrow and spleen, and splenomegaly. Deficiency of SMS1, but not SMS2, prevented SM production and enhanced phosphatidylserine (PS) externalization on the plasma membranes of platelets and megakaryocytes. Consequently, SMS1-KO platelets were excessively cleared by macrophages in the spleen. Multimer formation in the plasma membrane of TMEM16F, a known calcium (Ca2+)-activated nonselective ion channel and Ca2+-dependent PS scramblase, was enhanced; the result was PS externalization to outer leaflets through increased Ca2+ influx in immortalized mouse embryonic fibroblasts established from SMS1-KO mice (SMS1-KO tMEFs), as seen with SMS1-KO platelets. Thus, SMS1 deficiency changed the TMEM16F distribution on the membrane microdomain, regulating Ca2+ influx-dependent PS exposure. SMS1-KO tMEFs in which TMEM16F was knocked out by using the CRISPR/Cas9 system lacked both the Ca2+ influx and excess PS exposure seen in SMS1-KO tMEFs. Therefore, SM depletion on platelet membrane microdomains due to SMS1 deficiency enhanced PS externalization via a Ca2+ influx through TMEM16F activation, leading to elevated platelet clearance and causing hemostasis dysfunction through thrombocytopenia. Our current findings show that the SM-rich microdomain generated by SMS1 is a potent regulator of thrombocytopenia through TMEM16F, suggesting that its dysfunction may be a novel additional mechanism of ITP.

Deficiency of sphingomyelin synthase 2 prolongs survival by the inhibition of lymphoma infiltration through ICAM-1 reduction.

The tumor microenvironment (TME) formation involving host cells and cancer cells through cell adhesion molecules (CAMs) is essential for the multiple steps of cancer metastasis and growth. Sphingomyelin synthase 2 (SMS2) is involved in inflammatory diseases such as obesity and diabetes mellitus by regulation of the SM/ceramide balance. However, the involvement of SMS2 in TME formation and metastasis is largely unknown. Here, we report that SMS2-deficient (SMS2-KO) mice show suppressed the EL4 cell infiltration to liver and prolonged survival time. ICAM-1 was identified as a candidate for the inhibition of TME formation in immortalized mouse embryonic fibroblasts (tMEFs) from mRNA array analysis for CAMs. Reduced SM/ceramide balance in SMS2-KO tMEFs suppressed the attachment of EL4 cells through transcriptional reduction of ICAM-1 by the inhibition of NF-κB activation. TNF-α-induced NF-κB activation and subsequent induction of ICAM-1 were suppressed in SMS2-KO tMEFs but restored by SMS2 re-introduction. In the EL4 cell infiltration mouse model, EL4 injection increased ICAM-1 expression in WT liver but not in SMS2-KO mouse liver. Therefore, inhibition of SMS2 may be a therapeutic target to suppress the infiltration of malignant lymphoma.

Bioproduction of prostaglandins in a transgenic liverwort, Marchantia polymorpha.

Prostaglandins are biologically active substances used in a wide range of medical treatments. Prostaglandins have been supplied mainly by chemical synthesis; nevertheless, the high cost of prostaglandin production remains a factor. To lower the cost of prostaglandin production, we attempted to produce prostaglandins using a liverwort, Marchantia polymorpha L., which accumulates arachidonic acid, which is known as a substrate of prostaglandins. Here we report the first bioproduction of prostaglandins in plant species by introducing a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla into the liverwort. The transgenic liverworts accumulated prostaglandin F2α, prostaglandin E2 and prostaglandin D2 which were not detected in the wild-type liverwort. Moreover, we succeeded in drastically increasing the bioproduction of prostaglandins using an in vitro reaction system with the extracts of transgenic liverworts.

The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells.

To express a foreign gene in plants effectively, a good expression system is required. Here we describe the identification of a transcriptional terminator that supports increased levels of expression. The terminators of several Arabidopsis genes were examined in transfected Arabidopsis T87 protoplasts. The heat shock protein 18.2 (HSP) terminator was the most effective in supporting increased levels of expression. The HSP terminator increases mRNA levels of both transiently and stably expressed transgenes approximately 2-fold more than the NOS (nopaline synthase) terminator. When combined with the HSP terminator, a translational enhancer increased gene expression levels approximately 60- to 100-fold in transgenic plants.

Mapping and characterization of DNase I hypersensitive sites in Arabidopsis chromatin.

Recent genome-wide analyses of yeast and human chromatin revealed the widespread prevalence of DNase I hypersensitive sites (DNase I HSs) at gene regulatory regions with possible roles in eukaryotic gene regulation. The presence of DNase I HSs in plants has been described for only a few genes, and we analyzed the chromatin structure of an 80 kb genomic region containing 30 variably expressed genes by DNase I sensitivity assay at 500 bp resolution in Arabidopsis. Distinct DNase I HSs were found at the 5' and/or 3' ends of most genes irrespective of their expression levels. Further analysis of well-characterized genes showed that the DNase I HSs occurred near cis-regulatory elements in the promoters of these genes. Upon transcriptional activation of a heat-inducible gene, the DNase I HS was extended into the vicinity of a cis-element and adjacent TATA element in the promoter. Concomitant with this change in DNase I HS, histones were acetylated, removed from the promoter, and a transcription activator bound to this cis-element. These results suggest that the DNase I HSs participate in the transcriptional regulation of Arabidopsis genes by enhancing the access of chromatin remodeling factors and/or transcription factors to their target sites as seen in yeast and human chromatin.

Expression of randomly integrated single complete copy transgenes does not vary in Arabidopsis thaliana.

The high variability of transgene expression is frequently observed in independent transgenic lines. Variability of transgene expression has been attributed to several factors, including differences in chromosome position, repeat sequences and copy number. The eukaryotic genome, with a heterogeneous chromatin structure, is not homogeneous for transcriptional activity. Chromatin structure at the site of integration can affect transgene expression; this phenomenon is called the position effect. In this study, we investigated whether position effects confer variability of transgene expression in Arabidopsis thaliana. We analyzed the expression of randomly integrated single 'complete' (intact, non-truncated, non-rearranged) copy transgenes in A. thaliana. Ten independent lines containing single complete copies of the transgene located at different chromosome positions showed very similar levels of transgene expression, and variability of transgene expression was not observed. This result indicates that position effects may not generally be a major cause of variability of transgene expression in A. thaliana.