We have previously shown that isolated islets embedded in type 1 collagen gel in the presence of a defined medium undergo transdifferentiation within 96 h to duct epithelial structures. The aim of this study was to identify the factors implicated in this process. Freshly isolated canine islets were embedded in type 1 collagen gel, Matrigel or agarose for up to 120 h and cultured in (i) Dulbecco's modified Eagle's medium (DMEM)/F12 plus cholera toxin (CT), (ii) medium CMRL1066 plus CT, (iii) CMRL1066 plus forskolin and (iv) CMRL1066 alone. At 16 h, intracellular levels of cAMP (fmol/10 3 islets) were increased in groups i-iii (642 17, 338 48, 1128 221) compared with group iv (106 19, P<0·01). Epithelial differentiation correlated with the total amount of intracellular cAMP measured over 120 h. Islet-epithelial transformation during the initial 36 h was associated with a wave of apoptosis which was followed by a wave of cell proliferation. During epithelial differentiation there was a progressive loss of all islet hormones and the concomitant expression of cytoskeletal proteins characteristic of duct epithelial cells. Islets in collagen and Matrigel demonstrated high rates of epithelial differentiation (63 2% and 71 4% respectively) compared with those in agarose gel (0 0%, P<0·001). Islets suspended in DMEM/F12 plus CT supplemented with soluble laminin or fibronectin did not undergo transformation. Prior incubation of freshly isolated islets with an integrin-binding arginine-glycineaspartate motif-presenting synthetic peptide also reduced islet transformation. These studies confirm the biological potential of islets of Langerhans to differentiate to duct epithelial structures. cAMP-mediated signal transduction and an appropriate integrin-matrix interaction are necessary for this process to proceed.
The expression of tetraspanin CD9 was found on blastocysts in mice and endometrium epithelial cells in human and bovine. However, it remains unknown how CD9 is involved in the precise dialogue between embryo and uterus during early pregnancy. This study was designed to investigate the functional roles of CD9 in the embryo implantation with monoclonal antibody against CD9 protein (anti-CD9 mAb) and antisense oligonucleotide against CD9 gene (AS-CD9). Our results showed that intrauterine injection of anti-CD9 mAb on day 4 of pregnancy significantly increased the number of embryos implanted (7·24±0·39 versus 4·04±0·38). In vitro, anti-CD9 mAb or AS-CD9 significantly enhanced embryo-outgrowth ability on the monolayer of uterus epithelial cells in a dose-dependent manner. However, the attachment of blastocysts to epithelial cells was unaffected. Furthermore, we found that anti-CD9 mAb or AS-CD9 stimulated matrix metalloproteinase 2 (MMP-2) production of blastocysts on Fibronectin. LY294002, a specific inhibitor of phosphoinositide 3-kinase, was able to counteract the effect of anti-CD9 mAb and AS-CD9 on outgrowth ability and production of MMP-2. Our results indicated that CD9 played a role of inhibiting embryo implantation. CD9 was able to impair embryo invasion and the production of MMP-2 through the phosphoinositide 3-kinase signaling pathway.
Hepatocellular carcinoma (HCC) is one of the most difficult cancer disease for diagnosis and treatment, with a low survival rate and high recurrence rate and mortality. Nuclear factor of activated T-cells 5 (NFAT5) is mediated by osmolality and proved to be a carcinogenic gene in some tumor. However in our study we considered NFAT5 as tumor suppressor of HCC. RT-qPCR was performed for NFAT5 expression in tumor tissues. NaCl was applied to make hyperosmotic treatment. We knockdowned and overexpressed NFAT5 to investigate its role in HCC. FCM was used for apoptosis assay. Transwell and scratch assay is proceeded for invasion.NFAT5 is downregulated in HCC tissue and cell lines, besides, upregulated by hyperosmolality. NFAT5 promotes apoptosis by regulating PARP-1,BAX/BCL2 while inhibits invasion through EMT-related protein claudin-1 and fibronectin. Hyperosmolality is also a protective factor for HCC. We considered hyperosmolality exhibited his protective effect by inducing NFAT5.In a word, NFAT5 inhibits invasion and promotes apoptosis in HCC, associated with osmolality.
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