Wickerhamomyces ciferrii (W.c), an unconventional heterothallic yeast species, is renowned for its high production of tetraacetyl phytosphingosine (TAPS). Due to its excellent performance in TAPS production, this study aimed to construct a genetic operating system of W.c to enhance the production of TAPS and to screen high-yielding strains by mutagenesis and genetic engineering, thus laying the foundation for further development of industrial production of sphingolipid metabolites. In this study, we selected two autonomous replication elements (CEN, 2μ) and mined 11 endogenous promoter elements to establish a genetic operating system in W. ciferrii. The overexpression of Syr2 and Lcb2 in the sphingolipid metabolism pathway significantly increased the production of TAPS. Meanwhile, we established a method for the identification of haploid mating types of W. ciferrii by combining RT-PCR and flow cytometry. Five strains of W. ciferrii with different mating types constructed from the standard diploid W. ciferrii ATCC 14091 were screened out. A-type haploid W.c 140 showcased the highest production of TAPS with a yield of 4.74 mg/g and a titer of 32.61 mg/L. Mutant strains W.c 140-A9 and W.c 140-A11 were induced by atmospheric pressure room temperature plasma mutagenesis. The recombinant strains W.c 140 OELcb2 and W.c 140 OESyr2 with overexpression were constructed with the genetic operating system established in this study. The TAPS yields of the mutant strains increased by 61.39% and 67.09%, respectively, compared with that of starting strain W.c 140. The recombinant strains cultured in the LCBNB medium achieved yields of 10.60 mg/g and 12.14 mg/g, respectively, representing 2.24 and 2.56 times of that in strain W.c 140. Moreover, the yields of the two recombinant strains were significantly higher than that of the diploid strain ATCC 14091. The genetic operating system and the haploid strain W.c 140 established in this study provide a basis for the subsequent establishment of genetic engineering tools for W. ciferrii.
Sphingosine/genetics* ; Saccharomycetales/metabolism* ; Genetic Engineering/methods* ; Promoter Regions, Genetic ; Metabolic Engineering/methods* ; Fungal Proteins/genetics*

BACKGROUND: Endothelial cells play a key role in the cytokine storm caused by influenza A virus. MicroRNA-155 (miR-155) is an important regulator in inflammation. Its role in the inflammatory response to influenza A infection, however, has yet to be elucidated. In this study, we explored the role as well as the underlying mechanism of miR-155 in the cytokine production in influenza A-infected endothelial cells. METHODS: Human pulmonary microvascular endothelial cells (HPMECs) were infected with the influenza A virus strain H1N1. The efficiency of H1N1 infection was confirmed by immunofluorescence. The expression levels of proinflammatory cytokines and miR-155 were determined using real-time polymerase chain reaction. A dual-luciferase reporter assay characterized the interaction between miR-155 and sphingosine-1-phosphate receptor 1 (S1PR1). Changes in the target protein levels were determined using Western blot analysis. RESULTS: MiR-155 was elevated in response to the H1N1 infection in HPMECs (24 h post-infection vs. 0 h post-infection, 3.875 ± 0.062 vs. 1.043 ± 0.013, P = 0.001). Over-expression of miR-155 enhanced inflammatory cytokine production (miR-155 mimic vs. negative control, all P < 0.05 in regard of cytokine levels) and activation of nuclear factor kappa B in infected HPMECs (miR-155 mimic vs. negative control, P = 0.004), and down-regulation of miR-155 had the opposite effect. In addition, S1PR1 was a direct target of miR-155 in the HPMECs. Inhibition of miR-155 enhanced the expression of the S1PR1 protein. Down-regulation of S1PR1 decreased the inhibitory effect of the miR-155 blockade on H1N1-induced cytokine production and nuclear factor kappa B activation in HPMECs. CONCLUSION MiR-155 maybe modulate influenza A-induced inflammatory response by targeting S1PR1.
Down-Regulation ; Endothelial Cells ; Humans ; Influenza A Virus, H1N1 Subtype/genetics* ; Influenza A virus ; Influenza, Human/genetics* ; MicroRNAs/genetics* ; Sphingosine-1-Phosphate Receptors

OBJECTIVE: LASS2/TMSG1 gene is a novel tumor metastasis suppressor gene cloned from human prostate cancer cell line PC-3M in 1999 by Department of Pathology,Peking University of Basic Medical Sciences. It was found out that protein encoded by LASS2/TMSG1 could interact with the c subunit of vacuolar-ATPase (ATP6V0C). In this study, we explored the effect of LASS2/TMSG1 and its mutants on proliferation, migration and invasion of human prostate cancer cells and its molecular mechanism. METHODS: We constructed four LASS2/TMSG1 mutants and stably transfected the variants to human prostate cancer cell line PC-3M-1E8 cell with high metastatic potential. The stable transfectants were identified by qPCR and Western blot through analyzing the expression of LASS2/TMSG1 and ATP6V0C, the cell biology functions of LASS2/TMSG1 and its four mutants were studied using growth curve,MTT assay, soft agar colony formation assay, wound migration assay, Matrigel invasion study and flow cytometry. Furthermore, immunofluorescence was used to analysis the interaction of LASS2/ TMSG1 mutants and ATP6V0C. RESULTS: LASS2/TMSG1 mRNA and protein in LASS2/TMSG1 group and Mut1-Mut4 groups were higher than that in Vector group; Western blot showed that ATP6V0C protein in LASS2/TMSG1 wild group was lower than that in Vector group, but ATP6V0C protein in LASS2/TMSG1 S248A group was obviously higher than that in Vector group. MTT test and growth curve assay showed growth ability in LASS2/TMSG1 S248A group was increasing compared with other groups from day 5. Soft Agar colony formation experiment showed anchor independent growth ability in LASS2/TMSG1 S248A group was higher than those in the other groups (P<0.05), Cell migrations (from 35.3%±3.2% to 70.3%±3%) in LASS2/TMSG1 S248A group was increasing compared with LASS2/TMSG1 wild group (P<0.01), and more cells passed through Matrigel in LASS2/TMSG1 S248A group compared with LASS2/TMSG1 wild group (from 50±3.2 to 203±6.5, P<0.01), the apoptosis rate in LASS2/TMSG1 S248A group was obviously higher than that in LASS2/TMSG1 wild group (from 7% to 15.1%, P<0.05), and the G0/G1 ratio in LASS2/TMSG1 S248A group was obviously higher than that in LASS2/TMSG1 wild group (from 51.0% to 85.4%). Furthermore, double immunofluorescent staining observed the colocalization between ATP6V0C and LASS2/TMSG1 protein and its mutations, the expression of ATP6V0C in LASS2/TMSG1 S248A group increased significantly compared with the other groups. CONCLUSION LASS2/TMSG1 S248A promotes proliferation, migration and invasion of prostate cancer cells through increasing ATP6V0C expression, suggesting that aa248-250 is an important function site for LASS2/TMSG1 in invasion suppression of prostate cancer cells.
Beijing ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Humans ; Male ; Membrane Proteins/genetics* ; Mutation ; Neoplasm Invasiveness ; Prostatic Neoplasms/genetics* ; Sphingosine N-Acyltransferase/genetics* ; Transfection ; Tumor Suppressor Proteins/genetics* ; Vacuolar Proton-Translocating ATPases

Objective: To screen lentiviral vectors carrying siRNA which can specifically down-regulate the gene expression of the sphingosine-1-phosphate receptor 3 (S1PR3) in the corpus cavernosum smooth muscle (CCSM) cells of rats with spontaneous hypertension (SHT) and investigate the influence of the vectors on the signaling pathways of ROCK1, ROCK2 and eNOS in the CCSM cells of SHT rats. METHODS: Using the S1PR3 mRNA sequence of the rat as an interfering target, we designed and synthesized three pairs of siRNA sequences (siRNA1, 2 and 3) targeting S1PR3 and one pair of negative control, and then constructed and packaged them into lentiviral vectors. We cultured the CCSM cells of SHT and Wistar-Kyoto (WKY) rats in vitro and randomly divided them into groups A (SHT untransfected control), B (SHT transfected and carrying negative control virus), C (SHT transfected and carrying siRNA1 targeting S1PR3), D (SHT transfected and carrying siRNA2 targeting S1PR3), E (SHT transfected and carrying siRNA3 targeting S1PR3), and F (WKY untransfected control). With the multiplicity of infection (MOI) = 60, we transfected the CCSM cells of the SHT rats with the lentiviral vector and then determined the expression of the green fluorescent protein (GFP) as well as the mRNA and protein expressions of S1PR3, ROCK1, ROCK2 and eNOS in the CCSM cells of the SHT and WKY rats by RT-PCR and Western blot. RESULTS: Gene sequencing proved the successful construction of the lentiviral vector. The transfection efficiency of the CCSM cells of the rats was >80% in groups B, C, D and E. Compared with group A, the mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 exhibited no significant difference in group B but were remarkably decreased in groups C, D, E and F (P< 0.05), most significantly in group E, with the inhibition rates of the mRNA and protein expressions of S1PR3 of (34.2±2.9) and (77.7±4.7)%, those of ROCK1 of (33.3±1.4) and (51.1±7.3)%, and those of ROCK2 of (30.8±3.6) and (58.32±5.5)%, respectively. The mRNA and protein expressions of eNOS in group A showed no significant difference from those in groups B, C, D and E (P>0.05) but remarkably lower than those in group F (P< 0.05). Compared with group F, the mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 were not significantly different from those in group E (P>0.05) but markedly increased in groups A, B, C and D (P< 0.05), while those of eNOS remarkably decreased in groups A, B, C, D and E (P< 0.05). CONCLUSIONS The three constructed lentiviral vectors carrying siRNA targeting different loci of the S1PR3 gene could significantly inhibit the expression of S1P3 as well as RhoA/Rho kinase signaling pathways in the CCSM cells of SHT rats, and the vector carrying siRNA3 exhibited the highest inhibitory effect.
Animals ; Down-Regulation ; Gene Expression ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; Lentivirus ; genetics ; Male ; Myocytes, Smooth Muscle ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Penis ; metabolism ; RNA, Messenger ; RNA, Small Interfering ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Inbred WKY ; Receptors, Lysosphingolipid ; genetics ; metabolism ; Signal Transduction ; Sphingosine-1-Phosphate Receptors ; Transfection ; rho-Associated Kinases ; metabolism

OBJECTIVETo explore the effects of LASS2/TMSG1 silencing on the growth, invasion and metastasis of prostate carcinoma cells and to investigate the related molecular mechanisms.

METHODSLASS2/TMSG1 expression of human prostate carcinoma cell line with low metastatic potentiality (PC-3M-2B4 cells) was knocked down using DNA vector-based small interfering RNA (shRNA), followed by evaluations of tumor cell invasion and metastasis.

RESULTSA stable PC-3M-2B4 cell line with expression of LASS2/TMSG1-shRNA was successfully established. MTT assay showed PC-3M-2B4 cells exhibited a strong proliferation after transfection of LASS2/TMSG1-shRNA.LASS2/TMSG1-shRNA transfected clones demonstrated an increased clonogenicity by soft agar colony formation assay and a significant increase of tumor cell invasion by matrigel invasion study.Flow cytometry showed that after LASS2/TMSG1 gene silencing, the apoptotic rate of PC-3M-2B4 cell significantly decreased (P<0.01) without significant cell cycle change (P>0.05).Eight weeks after implantation into subcutaneous tissues in BAL B/c (nu+) mice, the size and weight of sh-LASS2/TMSG1 xenografts were significantly larger than those of the control group (P<0.05).Nuclear proliferation index of the subcutaneous tumor was also higher in the LASS2/TMSG1 shRNA group than those in the control group. Lymph node metastasis was observed in 5 of 6 mice of LASS2/TMSG1 shRNA group and only 1 of 6 of the control group. V-ATPase activity, activities of secreted MMP-2 and MMP-9 and extracellular hydrogen ion concentration were significantly increased in LASS2/TMSG1-shRNA group compared with the control group (P<0.05).

CONCLUSIONSilencing of LASS2/TMSG1 promotes the growth, invasion and metastasis of prostate cancer cells through up-regulation of V-ATPase activity, indicating that LASS2/TMSG1 is a tumor metastasis suppressor gene.

Animals ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Gene Silencing ; Humans ; Hydrogen-Ion Concentration ; Lymphatic Metastasis ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA, Small Interfering ; genetics ; Sphingosine N-Acyltransferase ; genetics ; metabolism ; Transfection ; Tumor Burden ; Tumor Suppressor Proteins ; genetics ; metabolism ; Up-Regulation ; Vacuolar Proton-Translocating ATPases ; metabolism

OBJECTIVETo investigate the effects of sphingosine kinase 1 (SphK1) on the proliferation, migration and invasion of human colon cancer LoVo cells, and to explore the related mechanisms.

METHODSHuman colon cancer LoVo cells were divided into three groups: phorbol 12-myristate 13-acetate (PMA) was used to induce the activation of SphK1 in the PMA group, N,N-dimethylsphingosine (DMS) used to suppress the activity of SphK1 in DMS group, and the cells treated with equal amount of 0.9 % NaCl instead of drugs served as the control group. The activity of SphK1 was assayed by autoradiography, the cell proliferation was assessed by MTT assay, cell migration and invasion were examined by Boyden chamber assay, concentrations of sICAM-1 and sVCAM-1 were assayed by ELISA, and RT-PCR and Western blot were used to evaluate the mRNA and protein expression in the cells.

RESULTSThe activity of SphK1 was efficiently induced by PMA and significantly suppressed by DMS. PMA induced cell proliferation in a time- and dose-dependent manner. On the contrast, DMS suppressed cell proliferation in a time- and dose-dependent manner. After treating with PMA, the number of migrating and invasing cells were increased to 143.36 ± 8.73 and 118.46 ± 6.25, significantly higher than those of the control group (75.48 ± 6.12 and 64.19 ± 5.36). After treating with DMS, the number of migrating and invasing cells were decreased to 38.57 ± 3.24 and 32.48 ± 4.27, significantly lower than those of the control group (P < 0.01). The relative expression levels of FAK, ICAM-1 and VCAM-1 mRNA in the PMA group were 0.82 ± 0.06, 0.74 ± 0.05 and 0.89 ± 0.09, and those in the DMS group were 0.23 ± 0.02, 0.26 ± 0.03 and 0.37 ± 0.04, with significant differences between the PMA, DMS and control groups (P < 0.01). Compared with the control group, the relative expression levels of FAK and p-FAK proteins in the PMA group (0.52 ± 0.06 and 0.51 ± 0.06) were significantly elevated, and those of the DMS group (0.20 ± 0.03 and 0.09 ± 0.02) were significantly decreased. In addition, the concentrations of sICAM-1 and sVCAM-1 were significantly elevated with the activation of SphK1. On the contrary, those of the DMS group were significantly reduced with the suppression of SphK1 (Both P < 0.01).

CONCLUSIONSSphK1 may enhance the proliferation, migration and invasion of colon cancer LoVo cells through activating FAK pathway and up-regulating the expression of ICAM-1 and VCAM-1.

Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; enzymology ; metabolism ; pathology ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Focal Adhesion Kinase 1 ; genetics ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Neoplasm Invasiveness ; Phosphorylation ; drug effects ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; Sphingosine ; analogs & derivatives ; pharmacology ; Tetradecanoylphorbol Acetate ; pharmacology ; Vascular Cell Adhesion Molecule-1 ; genetics ; metabolism

OBJECTIVETo investigate whether FTY720 inhibits rat mesangial proliferation and extracellular matrix expansion through suppression of transforming growth factor β1-connective tissue growth factor (TGFβ1-CTGF) pathway, and to explore experimental evidence for its effect on mesangial proliferative glomerulonephritis.

METHODSA rat model of anti-Thy-1 mesangial proliferative glomerulonephritis was established and FTY720 intervention was performed. Periphery blood lymphocyte count, urine protein excretion, glomerular mesangial proliferation, protein and gene expression of TGFβ1 and CTGF and extracellular matrix protein including fibronectin, laminin and collagen IV in isolated glomeruli were documented at 1, 3 and 7 days after injection of anti-Thy-1 antibody.

RESULTSThe model group developed proteinuria at 1, 3 and 7 days after injection of anti-Thy-1 antibody, which were significantly higher [(27.9 ± 7.3), (63.5 ± 18.8) and (52.4 ± 15.4)mg/d, respectively] than those in the control group [(8.4 ± 2.4), (8.4 ± 2.1) and (10.4 ± 3.2) mg/d; respectively, P < 0.01]. FTY720 intervention group showed significantly decreased proteinuria at 3 and 7 days after injection [(31.4 ± 7.0), (25.5 ± 7.7) mg/d, respectively] than model group (P < 0.01), although higher than the control group (P < 0.01). After intervention for 3 and 7 days, FTY720 significantly down-regulated both TGFβ1 and CTGF gene and protein expression in cultured glomeruli, and suppressed the production of glomerular extracellular matrix protein secretion, leading to attenuated mesangial cell proliferation and extracellular matrix expansion in rat anti-Thy-1 mesangial proliferative glomerulonephritis.

CONCLUSIONFTY720 significantly attenuates mesangial proliferation and extracellular matrix expansion through inhibition of TGFβ1-CTGF pathway in rat, and thus ameliorates the development of anti-Thy-1 mesangial proliferative glomerulonephritis.

Animals ; Cell Proliferation ; Connective Tissue Growth Factor ; genetics ; metabolism ; Down-Regulation ; Extracellular Matrix Proteins ; metabolism ; Fingolimod Hydrochloride ; Gene Expression ; Glomerular Mesangium ; metabolism ; pathology ; Glomerulonephritis, Membranoproliferative ; immunology ; metabolism ; pathology ; Immunosuppressive Agents ; pharmacology ; Isoantibodies ; immunology ; Kidney Glomerulus ; metabolism ; pathology ; Male ; Propylene Glycols ; pharmacology ; Proteinuria ; urine ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Sphingosine ; analogs & derivatives ; pharmacology ; Thy-1 Antigens ; immunology ; Transforming Growth Factor beta1 ; genetics ; metabolism

Higher levels of body fat are associated with an increased risk for development numerous adverse health conditions. FTY720 is an immune modulator and a synthetic analogue of sphingosine 1-phosphate (S1P), activated S1P receptors and is effective in experimental models of transplantation and autoimmunity. Whereas immune modulation by FTY720 has been extensively studied, other actions of FTY720 are not well understood. Here we describe a novel role of FTY720 in the prevention of obesity, involving the regulation of adipogenesis and lipolysis in vivo and in vitro. Male C57B/6J mice were fed a standard diet or a high fat diet (HFD) without or with FTY720 (0.04 mg/kg, twice a week) for 6 weeks. The HFD induced an accumulation of large adipocytes, down-regulation of phosphorylated AMP-activated protein kinase alpha (p-AMPKalpha) and Akt (p-Akt); down-regulation of hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL) and perilipin mRNA as well as up-regulation of phosphorylated HSL (p-HSL, Ser563) and glycogen synthase kinase 3 alpha/beta (p-GSK3alpha/beta). All these effects were blunted by FTY720 treatment, which inhibited adipogenesis and promoted lipolysis. Also, FTY720 significantly decreased lipid accumulation in maturing preadipocytes. FTY720 down-regulated the transcriptional levels of the PPARgamma, C/EBPalpha and adiponectin, which are markers of adipogenic differentiation. FTY720 significantly increased the release of glycerol and the expression of the HSL, ATGL and perilipin, which are regulators of lipolysis. These results show that FTY720 prevented obesity by modulating adipogenesis and lipolysis, and suggest that FTY720 is used for the treatment of obesity.
3T3-L1 Cells ; AMP-Activated Protein Kinases/metabolism ; Adipocytes/*drug effects/physiology ; Adipogenesis/drug effects ; Animals ; Anti-Obesity Agents/*pharmacology/therapeutic use ; Antigens, Differentiation/genetics/metabolism ; Carrier Proteins/genetics/metabolism ; Cell Size ; Diet, High-Fat/adverse effects ; Disease Models, Animal ; Enzyme Activation ; Gene Expression Regulation, Enzymologic/drug effects ; Glycogen Synthase Kinase 3/genetics/metabolism ; Lipase/genetics/metabolism ; Lipolysis/drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Obesity/etiology/metabolism/*prevention & control ; Phosphoproteins/genetics/metabolism ; Phosphorylation ; Propylene Glycols/*pharmacology/therapeutic use ; Protein Processing, Post-Translational ; Proto-Oncogene Proteins c-akt/metabolism ; Sphingosine/*analogs & derivatives/pharmacology/therapeutic use ; Sterol Esterase/metabolism

This study is to investigate the effects of ceramide on GSTA1 expression in Caco-2 cells. After being exposed to ceramide for a fixed time, GSTA1 protein expression was detected by Western blotting analysis; GSTA1 mRNA expression was detected by real time PCR; dual luciferase assay was used to analyze GSTA1 transcriptional activity and GSTA1 activity was determined toward androstanedione (AD) as substrate. The data showed that ceramide can significantly induce the expression of protein and GSTA1 mRNA, and increase transcriptional activity and enzyme activity of GSTA1. The results demonstrated that ceramide may increase resistance to chemotherapeutics in Caco-2 cells by up-regulating the expression of GSTA1.
Caco-2 Cells ; Enzyme Activation ; drug effects ; Glutathione Transferase ; genetics ; metabolism ; Humans ; RNA, Messenger ; metabolism ; Sphingosine ; analogs & derivatives ; pharmacology ; Transcriptional Activation ; drug effects ; Up-Regulation

OBJECTIVE: To investigate the variation of senescent endothelial function by regulating the sphingosine-1-phosphate receptor type 2 (S1P2) expression in cultured human umbilical vein endothelial cells (HUVECs). METHODS: The S1P2 receptor expression was regulated by transfecting the cDNA or shRNA of S1P2 in cultured HUVECs. The expression levels of S1P2 receptor in HUVECs were detected by RT-PCR and Western blot. EC chemotaxis was measured by the transwell migration assay. The wound healing assay was performed by a scratch wound model on EC monolayer. Matrigel morphogenesis assay was employed to assess the in vitro angiogenic responses. RESULTS: After up-regulating the S1P2 expression in young ECs, the S1P-stimulated formation of a tubular-like network in Matrigel was dramatically diminished in transfected ECs (P<0.05). Quantification of the wound healing assay showed that transfected ECs grew much slower than young ECs (P<0.05). The chemotactic capability was significantly decreased in transfected ECs (P<0.05). Furthermore, the senescent-associated impairments were revoked by the downregulation of S1P2 receptor in senescent HUVECs. CONCLUSION The impaired functions (chemotactic, wound-healing and morphogenetic responses) in senescent HUVECs in vitro are mediated by S1P2 receptor.
Cells, Cultured ; Cellular Senescence ; genetics ; Human Umbilical Vein Endothelial Cells ; cytology ; physiology ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptors, Lysosphingolipid ; genetics ; metabolism ; Sphingosine-1-Phosphate Receptors ; Transfection ; Up-Regulation