The role of vitamin D through SphK1/S1P in the regulation of MS progression.

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-ß, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-ß, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

Acetylshikonin induces autophagy-dependent apoptosis through the key LKB1-AMPK and PI3K/Akt-regulated mTOR signalling pathways in HL-60 cells.

Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti-inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK-induced cell death and the potential molecular mechanisms in human AML HL-60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL-60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S-phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho-Akt and p-p70S6K expression, while enhanced phospho-AMP-activated protein kinase (AMPK) and phospho-liver kinase B1(LKB1) expression. The suppression of ASK-induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK-induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis-related markers caspase-3 and caspase-9 and the activity of caspase-3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3-methyladenine (3-MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK-induced HL-60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt-regulated mTOR signalling pathways.

Induction of S-Phase Arrest in Human Glioma Cells by Selenocysteine, a Natural Selenium-Containing Agent Via Triggering Reactive Oxygen Species-Mediated DNA Damage and Modulating MAPKs and AKT Pathways.

Selenocysteine (SeC) a natural available selenoamino acid exhibits novel anticancer activities against human cancer cell lines. However, the growth inhibitory effect and mechanism of SeC in human glioma cells remain unclear. The present study reveals that SeC time- and dose-dependently inhibited U251 and U87 human glioma cells growth by induction of S-phase cell cycle arrest, followed by the marked decrease of cyclin A. SeC-induced S-phase arrest was achieved by inducing DNA damage through triggering generation of reactive oxygen species (ROS) and superoxide anion, with concomitant increase of TUNEL-positive cells and induction of p21waf1/Cip1 and p53. SeC treatment also caused the activation of p38MAPK, JNK and ERK, and inactivation of AKT. Four inhibitors of MAPKs and AKT pathways further confirmed their roles in SeC-induced S-phase arrest in human glioma cells. Our findings advance the understanding on the molecular mechanisms of SeC in human glioma management.

The involvement of chloroplast HSP100/ClpB in the acquired thermotolerance in tomato.

The chloroplast HSP100/ClpB is a newly documented member of the ClpB family, but little was known about its role in imparting thermotolerance to cells. A cDNA coding for a HSP100/ClpB homolog has been cloned from Lycopersicon esculentum and termed as Lehsp100/ClpB (the cDNA sequence of Lehsp100/ClpB has been submitted to the GenBank database under accession number: AB219939). The protein encoded by the cDNA was most similar to the putative chloroplast HSP100/ClpBs in higher plants and the ClpB from Cyanobacterium Synechococcus sp. A 97 kDa protein, which matched the predicted size of mature LeHSP100/ClpB, was immunologically detected in chloroplast isolated from heat-treated tomato plants. In addition, the fusion protein, combining the transit sequence of LeHSP100/ClpB and GFP, was found to be located in chloroplast based on the observations of fluorescent microscope images. These results indicated the chloroplast-localization of LeHSP100/ClpB. Both the transcript and the protein of Lehsp100/ClpB were not detected under normal growth conditions, but they were induced by increasingly higher temperatures. An antisense Lehsp100/ClpB cDNA fragment was introduced into the tomato by Agrobacterium-mediated transformation. Antisense lines exhibited an extreme repression of heat-induced expression of Lehsp100/ClpB. The levels of chloroplast HSP60 and small HSP in antisense lines were identical to those of the control plants. After plants preconditioned at 38 degrees C for 2 h were exposed to a lethal heat shock at 46 degrees C for 2 h, the antisense lines were greatly impaired and withered in 21 days of the recovery phase, whereas the untransformed control plants and the vector-transformed plants survived. Furthermore, chlorophyll fluorescence measurements showed that PS II in antisense lines were more susceptible to the thermal irreversible inactivation than the untransformed and vector-transformed control plants. This work provides the first example that induction of chloroplast LeHSP100/ClpB contributes to the acquisition of thermotolerance in higher plants.

[Molecular cloning of tomato LeHsp110/ClpB gene and its effect on the thermotolerance in plant].

The heat shock protein ClpB is a member of the Clp family and functions as molecular chaperones. ClpB is related to the acquired thermotolerance in organisms. A cDNA of 3144 bp was screened out of a tomato cDNA library. The polypeptide deduced from the longest ORF contains 980 amino acid residues, and was classified into HSP100/ClpB family based on the result of molecular phylogenesis analysis. Thus it was named as LeHSP110/ClpB according to its calculated molecular weight. LeHSP110/ClpB was characteristic of heat-inducibility but no constitutive expression, and was demonstrated to locate in chloroplastic stroma. An antisense cDNA fragment of LeHsp110/ClpB under the control of CaMV 35S promoter was introduced into tomato by Agrobacterium tumefactions-mediated method. At high temperature, the mRNA levels of LeHsp110/ClpB in antisense transgenic plants were lower than those in control plants. The PS II of transgenic plants is more sensitive to high temperature than that of control plants according to data of Fv/Fm. These results clearly showed that HSP110/ClpB plays an important role in thermotolerance of high plants.

Cloning and molecular characteristic of the metalloprotease gene LeftsH6 from tomato.

The full-length 2213-bp ftsH (filamentation temperature-sensitive H) cDNA was cloned from the cDNA library of heat-shocked tomato leaves. According to an open reading frame of 2019-bp, the deduced protein precursor was predicted to target chloroplast. The putative AAA (ATPases associated with diverse cellular activities) domain and the Zn(2+)-binding domain, characteristic of FtsH metalloproteases family, were found in the FtsH-like protein. Most similar to the FtsH6 of Arabidopsis thaliana, the tomato ftsH-like gene was named as Lycopersicon esculentum filamentation temperature-sensitive H6 (LeftsH6). Purified FtsH degraded casein but not BSA in vitro, whereas a FtsH mutant with the Glu(472) in the zinc-binding motif replaced by Gln had lost the protease activity. A single copy of LeftsH6 was detected in tomato genome by Southern blot analysis. Northern and Western blot analyses revealed consistently the heat-inducible character of the LeftsH6 gene. No LeftsH6 expression was detected after cold, salt, drought or light stress. The results provided the first experimental evidence of the existence of heat-inducible ftsH gene in higher plants.

Differential regulation of Lehsp23.8 in tomato plants: Analysis of a multiple stress-inducible promoter.

Small heat shock proteins (sHSPs) are the major family of HSP induced by heat stress in plants. In this report, an approximately 1.9kb of Lehsp23.8 5'-flanking sequence was isolated from tomato genome. By using the ß-glucuronidase (GUS) reporter gene system, the developmental and tissue specific expression of the gus gene controlled by the Lehsp23.8 promoter was characterized in transgenic tomato plants. Strong GUS staining was detected in the roots, leaves, flowers, fruits and germinated seeds after heat shock. The heat-induced GUS activity was different in the floral tissues at various developmental stages. Fluorometric GUS assay showed that the heat-induced GUS activity was higher in the pericarp than in the placenta, and it was the lowest in the locular gel. The heat-shock induction of the Lehsp23.8 promoter depended on the different stages of fruit development. The optimal heat-shock temperatures leading to the maximal GUS activity in the pericarp of green, breaker, pink and red fruits were 42, 36, 39 and 39°C, respectively. The heat-induced GUS activity in tomato fruits increased gradually within 48h of treatment and weakened during tomato fruit ripening. Obvious GUS activities under cold, exogenous ABA and heavy metal (Cd(2+), Cu(2+), Pb(2+) or Zn(2+)) stress conditions were also detected. These results show that the Lehsp23.8 promoter is characterized as strongly heat-inducible and multiple-stress responsive.