Prebiotics can attenuate decreased intestinal immunity and enhanced oxidative stress, in addition to reduced intestinal peristalsis and of the constipated rats.

DCBLD2 is a neuropilin-like transmembrane protein that is up-regulated during arterial remodeling in humans, rats, and mice. Activation of PDGFR-β via PDGF triggers receptor phosphorylation and endocytosis. Subsequent activation of downstream signals leads to the stimulation of phenotypic conversion of VSMCs and arterial wall proliferation, which are common pathological changes in vascular remodeling diseases such as atherosclerosis, hypertension, and restenosis after angioplasty. In this study, we hypothesized that DCBLD2 regulates neointimal hyperplasia through the regulation of PDGFR-β endocytosis of vascular smooth muscle cells (VSMCs) through Caveolin-1 (Cav-1). Compared with wildtype (WT) mice or control littermate mice, the germline or VSMC conditional deletion of the Dcbld2 gene resulted in a significant increase in the thickness of the tunica media in the carotid artery ligation. To elucidate the underlying molecular mechanisms, VSMCs were isolated from the aorta of WT or Dcbld2 −/− mice and were stimulated with PDGF. Western blotting assays demonstrated that Dcbld2 deletion increased the PDGF signaling pathway. Biotin labeling test and membrane-cytosol separation test showed that after DCBLD2 was knocked down or knocked out, the level of PDGFR-β on the cell membrane was significantly reduced, while the amount of PDGFR-β in the cytoplasm increased. Coimmunoprecipitation experiments showed that after DCBLD2 gene knock-out, the binding of PDGFR-β and Cav-1 in the cytoplasm significantly increased. Double immunofluorescence staining showed that PDGFR-β accumulated Cav-1/ lysosomes earlier than for control cells, which indicated that DCBLD2 gene knock-down or deletion accelerated the endocytosis of PDGF-induced PDGFR-β in VSMCs. In order to confirm that DCBLD2 affects the relationship between

On the basis of their characteristics, we presume that developmental stage-specific hepatocytes should have the ability to induce maturation of hepatoma cells. A regulatory circuit formed by hepatocyte nuclear factor (HNF)-4α, HNF-1α, HNF-6 and the upstream stimulatory factor (USF-1) play a key role in the maturation of embryonic hepatocytes; however, it is unclear whether the regulatory circuit mediates the embryonic induction of hepatoma cell maturation. In this study, 12.5-d to 15.5-d mouse embryonic hepatocytes or their medium were used to coculture or treat HepG2 cells, and the induced maturation was evaluated in vitro and in vivo. In the induced HepG2 cells, the components of the regulatory circuit were detected, their cross-regulation was evaluated and HNF-4α RNA interference was performed. We found that 13.5-d to 14.5-d embryonic hepatocytes could induce HepG2 cell maturation, demonstrated by morphological changes, increased maturation markers and decreased c-Myc and α-fetoprotein (AFP) in vitro. The majority of HepG2 tumors were eliminated by 13.5-d embryonic induction in vivo. All components of the regulatory circuit were upregulated and the binding of HNF-4α, HNF-1α, HNF-6 and USF-1 to their target sites was promoted to rebuild the regulatory circuit in the induced HepG2 cells. Moreover, RNA interference targeting HNF-4α, which is the core of the regulatory circuit, attenuated the induced maturation of HepG2 cells with downregulation of the regulatory circuit. These results revealed that developmental stage-specific hepatocytes could induce the maturation of HepG2 cells by rebuilding the regulatory circuit. is believed to be a controller and marker of mature hepatocytes (27,28). As a downstream target of HNF-4α, HNF-1α is also essential for hepatocyte maturation (29,30). USF-1, which competes with the oncogene c-Myc for the E-box regulatory element, is another downstream target of HNF-4α that plays crucial roles in the maturation and formation of functional hepatocytes (31,32). USF-1 can activate HNF-6, which is another important transcription factor for hepatocyte maturation that has been found to regulate 33). Therefore, HNF-4α, HNF-1α, HNF-6 and USF-1 form a positive feedback loop that drives hepatocyte maturation in liver development ( Figure 1A). Because the transcription factor binding sites are generally believed to scatter within 10 kb upstream of the transcriptional start site (34), the 10-kb gene promoters were all analyzed. On the basis of previous reports (25), the predicted binding sites of HNF-4α HNF-1α, HNF-6 and USF-1 were identified, respectively ( Figure 1B). It remains unknown whether developmental stage-specific hepatocytes rebuild the regulatory circuit to induce the maturation of HepG2 cells.In this study, HepG2 cells were cocultured with mouse embryonic hepatocytes at gestation of 12.5-15.5 d, because the mouse hepatocytes differentiated at 12.5 d, and the liver structure became firmly established between 12 and 15 d of gestation (35)(36)(37). The induced maturation of HepG2 cel...

This study demonstrated that developmental stage-specific embryonic induction of HepG2 cell differentiation might help in understanding embryonic differentiation and oncogenesis.

ObjectiveUnwanted angiogenesis is involved in the progression of various malignant tumors and cardiovascular diseases, and the factors that regulate angiogenesis are potential therapeutic targets. We tested the hypothesis that DCBLD1 (Discoidin, CUB, and LCCL domain-containing protein 1) is a co-receptor of VEGFR-2 and modulates angiogenesis in endothelial cells(ECs).Approach and ResultsA carotid artery ligation model and retinal angiogenesis assay were used to study angiogenesis using globe knockout or EC-specific conditional DCBLD1 knockout micein vivo. Immunoblotting, immunofluorescence staining, plasma-membrane subfraction isolation, Co-immunoprecipitation and mass-spectrum assay were performed to clarify the molecular mechanisms.Loss of DCBLD1 impaired VEGF response and inhibited VEGF-induced EC proliferation and migration. DCBLD1 deletion interfered with adult and developmental angiogenesis. Mechanistically, DCBLD1 bound to VEGFR-2 and regulated the formation of VEGFR-2 complex with negative regulators: protein tyrosine phosphatases, E3 ubiquitin ligases(Nedd4 and c-Cbl), and also DCBLD1 knockdown promoted lysosome-mediated VEGFR-2 degradation in ECs.ConclusionsThese findings demonstrated the essential role of endothelial DCBLD1 in regulating VEGF signaling and provided evidence that DCBLD1 promotes VEGF-induced angiogenesis by limiting the dephosphorylation, ubiquitination, and lysosome degradation after VEGFR-2 endocytosis. We proposed that endothelial DCBLD1 is a potential therapeutic target for ischemic cardiovascular diseases by the modulation of angiogenesis through regulating of the VEGFR-2 endocytosis.