TGFBR3 supports anoikis through suppressing ATF4 signaling.

Epithelial morphogenesis and oncogenic transformation can cause loss of cell adhesion, and detached cells are eliminated by anoikis. Here, we reveal that transforming growth factor ß receptor 3 (TGFBR3) acts as an anoikis mediator through the coordination of activating transcription factor 4 (ATF4). In breast cancer tissues, TGFBR3 is progressively lost, but elevated TGFBR3 is associated with a histologic subtype characterized by cellular adhesion defects. Dissecting the impact of extracellular matrix (ECM) deprivation, we demonstrate that ECM loss promotes TGFBR3 expression, which in turn causes differentiation of cell aggregates, conferring a low-adhesion phenotype, and drives the intrinsic apoptotic pathway. We demonstrate that inhibition of TGFBR3 impairs epithelial anoikis by activating ATF4 signaling. These preclinical findings provide a rationale for therapeutic inhibition of ATF4 in the subgroup of breast cancer patients with low TGFBR3 expression.

Dapper1 attenuates hepatic gluconeogenesis and lipogenesis by activating PI3K/Akt signaling.

Studies have shown that hepatic insulin resistance, a disorder of glucose and lipid metabolism, plays a vital role in type 2 diabetes (T2D). To clarify the function of Dapper1 in glucose and lipid metabolism in the liver, we investigated the relationships between Dapper1 and adenosine triphosphate (ATP)- and Ca2+-mediated activation of PI3K/Akt. We observed a reduction in hepatic Dapper1 in db/db (mice that are homozygous for a spontaneous diabetes mutation) and HFD-induced diabetic mice with T2D. Hepatic overexpression of Dapper1 improved hyperglycemia, insulin resistance, and fatty liver. It also increased Akt (pAkt) signaling and repressed both gluconeogenesis and lipogenesis. Conversely, Ad-shDapper1-induced knockdown of hepatic Dapper1 promoted gluconeogenesis and lipogenesis. Furthermore, Dapper1 activated PI3K p110α/Akt in an insulin-independent manner by inducing ATP production and secretion in vitro. Blockade of P2 ATP receptors, the downstream phospholipase C (PLC), or the inositol triphosphate receptor (IP3R all reduced the Dapper1-induced increase in cytosolic free calcium and Dapper1-mediated PI3K/Akt activation, as did removal of calcium in the medium. In conclusion, Dapper1 attenuates hepatic gluconeogenesis and lipogenesis in T2D.

[Subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 protein].

OBJECTIVE: To study the subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) protein in endothelial cells. METHODS: Human umbilical vein endothelial cell strain ECV304 were cultured in vitro. The fusion protein of enhanced green fluorescent protein (EGFP)-EOLA1 expressing plasmid was constructed. Empty plasmid with EGFP at N side (pEGFP-N2) and fusion protein expressing plasmid EGFP-EOLA1 was respectively transfected into ECV304 cells with liposome. After being cultured for 48 hours, the expression levels of EGFP and fusion protein EGFP-EOLA1 in cells were detected with Western blot. The subcellular localization of EOLA1 protein was detected by laser scanning confocal microscope and immunoelectron microscopy. RESULTS: The EGFP-EOLA1 coexpression plasmid was verified to be successfully constructed by enzyme cutting and gene sequencing. The fusion protein of EGFP-EOLA1 was observed to express in transfected cells through Western blot. Green fluorescence scattered all over the ECV304 cells transfected with empty plasmid and cells transfected with fusion protein expressing plasmid, and it gathered obviously in the nuclei in the latter cells. Immune deposits were observed in the matrix of cells transfected with fusion protein expressing plasmid but not in the cells transfected with empty plasmid. CONCLUSIONS: EOLA1 protein is localized in the nucleus and the matrix of ECV304 cell, and it plays its role as a signal transduction factor.

[The effect of inhibiting EOLA1 expression in ECV304 cells].

OBJECTIVE: To study the effect of inhibiting the expression of endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) on proliferation of human umbilical vein endothelial cell line ECV304. METHODS: After constructing and transfecting EGFP-EOLA1 fusion protein expressive vector into ECV304 cells, the transfected cells was cultured in M199 containing G418 for 5 weeks to screen the cell line stable expression EGFP-EOLA1 fusion protein. Oligonucleotides targeting EOLA1 at different sites were synthesized and inserted into pSinencer3.1/H1 vector. Then, the recombinant vector was transfected into the cultured ECV304 cells and the inhibiting effect to target gene EOLA1 was investigated by observing the green fluorescence in transfected cells under inverted fluorescent microscope and by Western blot assay. The proliferation of ECV304 cells was numbered when the expression of EOLA1 in ECV304 cells was inhibited by RNA interference. RESULTS: The ECV304 cell line stably expressing EGFP-EOLA1 fusion protein was constructed and the siEOLA1 interfere vectors can knock down EOLA1 gene expression specially. When blocking the expression of EOLA1 in ECV304 cells,the proliferation of cells slowed down. CONCLUSION: EOLA1 maybe has a role on the proliferation of cells.

[Purification of human endothelial overexpressed lipopolysaccharide-associated factor 1 protein].

OBJECTIVE: To investigate the feasibility of obtaining of a highly pure protein of human endothelial overexpressed lipopolysaccharide-associated factor 1 (EOLA1) with metal chelation chromatography. METHODS: Inclusion bodies of the E. coli transformed with EOLA1 gene were extracted and washed with BugBuster Protein Extraction Reagent. The primary purified products were purified by His. Bind Resin Chromatography under denaturing condition and dialyzed for renaturation, and then were analyzed with SDS-PAGE, Western blotting and peptide mass fingerprinting (PMF). RESULTS: EOLA1 was mainly expressed in E. coli as insoluble inclusion bodies. The protein content in the primary extracted inclusion bodies accounted for over 75%, and it accounted for more than 90% after chromatography and renaturation. It was indicated by PMF that the targeted protein peptide overlaid many of designed protein peptide. CONCLUSION: The method of EOLA1 protein purification and renaturation was convenient and efficient, and by this method sufficient amount of highly pure EOLA1 protein could be obtained for the preparation of EOLA1 monoclonal antibody and for the study of its gene function.

[Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation].

OBJECTIVE: To amplify the full-length cDNA and characterize the structure and biological function of a novel expression sequence tag ST55 (GenBank Accession No. BM121646). METHODS: Rapid amplification of cDNA ends was used to clone the full-length of cDNA of ST55 in this study. Then, its tissue distribution was checked by Northern blots, and the associated protein was screened by GAL 4-based yeast two-hybrid. The effect of stable transfection of the cDNA on cell proliferation was evaluated in ECV304 cells. RESULTS: A full-length 1404 bp cDNA was cloned, and it was accepted as a novel human mRNA by GenBank (No. AY074889), named endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1). Bioinformatic analysis found that the EOLA1 encoded 158 amino acids, 17.89 kDa protein, and mapped to chromosome Xq27.4 with 5 exons. EOLA1 expressed in different human normal tissues and cancer cell lines. Using the EOLA1 cDNA as bait, we performed a yeast two-hybrid screening of a human liver cDNA library and identified metallothionein 2A (MT2A) as associated protein. The interaction between EOLA1 and MT2A was confirmed by co-immunoprecipitation experiments. Stable transfection of EOLA1 was noted to stimulate ECV304 cell proliferation (P < 0.05). CONCLUSION: The findings suggest that EOLA1 is a novel gene and the interaction of EOLA1 and MT2A may play an important role in cell protection in inflammation reaction.

[Establishment of human endothelial-overexpressed lipopolysaccharide-associated factor 1 compelling expression model and its effects on the proliferation of ECV304 cells].

OBJECTIVE: To design and construct the inducible expression vector of endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1), in order to establish EOLA1 compelling expression model, and to observe the effects of EOLA1 compelling expression on the proliferation of ECV304 cells. METHODS: Inducible overexpression vector pOPRSV I-EOLA1 was constructed by amplifying the open reading fragment of EOLA1 and subcloning it into the Not I site and Xho I site of pOPRSV I vector. After sequencing, the pOPRSV I-EOLA1 recombinant vector and pCMVLac I vector were co-transfected into ECV304 cells. The cells resistant to G418 and hygromycin were screened by G418 and hygromycin, so that stable transfected cell strain was obtained. The growth curve of cells with or without isopropyl-beta-D-thiogalactoside (IPTG) induction were graphed with cell counting. RESULTS: The inducible overexpressed EOLA1 vector was constructed successfully. The proliferation of the cells with EOLA1 compelling expression after induction of IPTG (44 +/- 17) x 10(4) was significantly higher than that without IPTG induction (27 +/- 11) x 10(4), (P < 0.01). CONCLUSION: Compelling expression of EOLA1 protein can enhance the proliferation of ECV304 cell.

Cloning of differentially expressed genes following lipopolysaccharide stimulation in human umbilical vein endothelial cells.

OBJECTIVE: To clone the differentially expressed genes in human umbilical vein endothelial cells (HUVEC) stimulated by lipopolysaccharide (LPS). METHODS: Two-directional (forward and backward) suppression subtractive hybridization (SSH) was performed on HUVEC cultured in either standard media or treated for 6 hours with LPS (100 ng/ml). To restrict the number of false-positive clones, colony dot hybridization was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced. RESULTS: These analyses have identified both novel and known genes whose expression is influenced by LPS. The known genes include a group related to proinflammatory events, a group related to cellular apoptosis and proliferation, a group related to protein synthesis and cytoskeletal rearrangment, and a group related to energy metabolism and signal transduction. CONCLUSIONS: SSH is a powerful technique of high sensitivity for the detection of differential gene expression in HUVEC stimulated by LPS.