Search

Homeobox containing 1 (HMBOX1) is essential for the survival of human umbilical vein endothelial cells (HUVECs). However, the regulatory mechanism of HMBOX1 expression is still unclear. We recently found that a small molecule 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (ABO) directly targeted annexin A7 (ANXA7) and inhibited its GTPase activity. In addition, both HMBOX1 and ANXA7 participated in the autophagy and apoptosis of HUVECs. But, their relationship in the regulation of HMBOX1 expression is unknown. In this study, we found that ABO could elevate HMBOX1 at translation level through inhibiting ANXA7 GTPase activity. ABO failed to increase HMBOX1 protein level in ANXA7-deficient HUVECs. TGFB2 overlapping transcript 1 (TGFB2-OT1) that was increased by ABO facilitated HMBOX1 expression by increasing La-related protein 1 (LARP1) expression. Furthermore, the protein level of HMBOX1 was decreased under oxidized low-density lipoprotein (oxLDL) treatment in HUVECs and in the aortic endothelium of apolipoprotein E-deficient (apoE -/- ) mice, which could be reversed by ABO in vitro and in vivo. In conclusion, ANXA7 was an endogenous regulator of HMBOX1, and ABO promoted HMBOX1 translation by inhibiting ANXA7 GTPase activity and enhancing TGFB2-OT1 expression. Besides, our data suggested that HMBOX1 might be a novel diagnostic marker and therapeutic target of atherosclerosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

T-cell intracellular antigen-1 (TIA1) is a DNA/RNA binding protein broadly expressed in eukaryotic cells, participating in multiple aspects of cellular metabolism. TIA1 phosphorylation was related with cell apoptosis and its RNA binding activity, however, the regulator and other functions of TIA1 phosphorylation were very little known. To find the modulator of TIA1 phosphorylation, we performed yeast two-hybrid screening and identified annexin A7 (ANXA7) as an interaction protein of TIA1. Recent study showed that a small molecule ABO could directly target ANXA7 and inhibit ANXA7 activity and its targets' phosphorylation. As a GTPase, ANXA7 was speculated to modulate TIA1 phosphorylation. Our results showed that ABO treatment promoted the interaction between TIA1 and ANXA7, and then greatly inhibited phosphorylation of TIA1 in HUVECs. Further results showed that ABO-increased interaction between ANXA7 and TIA1 significantly promoted the processing of a pro-autophagic factor FLJ11812 and the expression of ATG13. Moreover, we found that ABO increased TIA1 protein level, co-localization of ANXA7 and TIA1, and ATG13 expression in the aortic endothelium of apoE(-/-) mice. These data highlighted the new role of TIA1 phosphorylation in autophagy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We recently found that both annexin A7 and integrin β4 were involved in autophagy of vascular endothelial cells. But, their relation is not clear. In this study, we addressed this question by using a small molecule ABO that promoted autophagy by targeting annexin A7. The results showed that knockdown of integrin β4 partly inhibited ABO-induced autophagy in vascular endothelial cells. Furthermore, in HEK293 cells that express integrin β4 too low to detect by western blot, ABO could not induce autophagy. If integrin β4 was overexpressed in HEK293 cells, ABO could evoke autophagy. On the other hand, knockdown of annexin A7 also blocked ABO-induced autophagy although the level of integrin β4 was elevated. Moreover, by co-immunoprecipitation, we identified the interaction of integrin β4 and annexin A7, and found that ABO could modulate the interaction, at the same time, the phosphorylation of Y-1494 in integrin β4 cytoplasmic domain was inhibited significantly in vitro and in vivo. Hence, by identifying the interaction between integrin β4 and annexin A7, we demonstrated that both annexin A7 and integrin β4 were essential for small molecule ABO-induced autophagy and targeting annexin A7 by ABO could modulate integrin β4 phosphorylation, while Y-1494 phosphorylation of integrin β4 may negatively regulate autophagy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Although bone marrow stromal cells (BMSCs) can differentiate into neuron-like cells, the mechanisms underlying neuronal differentiation are not well understood. We recently found that inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC) by its inhibitor D609 promoted BMSCs' differentiation into cholinergic neuron-like cells. Using the effective small molecule D609 and gene microarray technology, we investigated the change of gene expression profile to identify key mediators involved in the neuronal differentiation. We selected heat shock protein 70 (Hsp70) and transcription factor B-cell translocation gene 2 (Btg2) that were maximally up-regulated for further study. We found that functional suppression of Hsp70 blocked D609-induced increase of Btg2 expression and cholinergic neuronal differentiation of BMSCs. These results demonstrated that Hsp70 was the pivotal factor in PC-PLC-medicated neuronal differentiation of BMSCs, and Btg2 might be its downstream target. Our findings provide new clues for controlling BMSCs' differentiation into cholinergic neuron-like cells and provide a putative strategy for neurodegenerative diseases therapies., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Lipopolysaccharide (LPS)-induced vascular endothelial cell (VEC) dysfunction is an important contributing factor in vascular diseases. Recently, we found that LPS impaired VEC by inducing autophagy. Our previous researches showed that a butyrolactone derivative, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) selectively protected VEC function. The objective of the present study is to investigate whether and how 3BDO inhibits LPS-induced VEC autophagic injury. Our results showed that LPS induced autophagy and led to increase of reactive oxygen species (ROS) and decrease of mitochondrial membrane potential (MMP) in Human umbilical vein vascular endothelial cells (HUVECs). Furthermore, LPS significantly increased p8 and p53 protein levels and the nuclear translocation of p53. All of these effects of LPS on HUVECs were strongly inhibited by 3BDO. Importantly, the ROS scavenger N-acetylcysteine (NAC) could inhibited LPS-induced autophagy and knockdown of p8 by RNA interference inhibited the autophagy, p53 protein level increase, the translocation of p53 into nuclei and the ROS level increase induced by LPS in HUVECs. The data suggested that 3BDO inhibited LPS-induced autophagy in HUVECs through inhibiting the ROS overproduction, the increase of p8 and p53 expression and the nuclear translocation of p53. Our findings provide a potential tool for understanding the mechanism underlying LPS-induced autophagy in HUVECs and open the door to a novel therapeutic drug for LPS-induced vascular diseases., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Neural stem cells are capable of differentiating into three major neural cell types, but the underlying molecular mechanisms remain unclear. Here, we investigated the mechanism by which integrin beta4 modulates mouse neural stem cell differentiation in vitro. Inhibition of endogenous integrin beta4 by RNA interference inhibited the cell differentiation and the expression of fibroblast growth factor receptor 2 but not fibroblast growth factor receptor 1 or fibroblast growth factor receptor 3. Overexpression of integrin beta4 in neural stem cells promoted neural stem cell differentiation. Furthermore, integrin beta4-induced differentiation of neural stem cells was attenuated by SU5402, the inhibitor of fibroblast growth factor receptors. Finally, we investigated the role of integrin beta4 in neural stem cell survival: knockdown of integrin beta4 did not affect survival or apoptosis of neural stem cells. These data provide evidence that integrin beta4 promotes differentiation of mouse neural stem cells in vitro possibly through fibroblast growth factor receptor 2.

Previously, we found that suppressing phosphatidylcholine-specific phospholipase C could induce neuronal differentiation of rat mesenchymal stem cells in the absence of serum and fibroblast growth factor. It is well known that basic fibroblast growth factor plays an important role in mesenchymal stem cell neuronal differentiation. In this study, our purpose was to understand the cooperation of phosphatidylcholine-specific phospholipase C and basic fibroblast growth factor in controlling mesenchymal stem cell neuronal differentiation. Our results showed that suppressing phosphatidylcholine-specific phospholipase C in the presence of basic fibroblast growth factor could induce cell neuronal differentiation and the viability of the differentiated cells was obviously increased. Furthermore, we found that the resting membrane potential of the differentiated cells gradually decreased, but the mitochondrial membrane potential rose with increasing treatment time and these characteristics were similar to cultured neurons from mouse embryo forebrains. To determine the possible mechanism by which this combination controls cell neuronal differentiation, we measured changes in the mitochondrial membrane potential and in the levels of reactive oxygen species. The results showed that both the mitochondrial membrane potential and reactive oxygen species levels decreased when basic fibroblast growth factor was added. The data suggested that lower phosphatidylcholine-specific phospholipase C activity was required for mesenchymal stem cell neuronal differentiation and basic fibroblast growth factor was necessary for maintaining the neuronal differentiation state. Moreover, basic fibroblast growth factor could contribute to rescuing the differentiated cells from death through decreasing overly high mitochondrial membrane potentials and reactive oxygen species levels.

Recently, the specific roles of integrin beta4 in the signaling networks that drive pathological angiogenesis and tumor progression have been revealed. Our previous study showed that integrin beta4 might be involved in neuron survival signal transduction. To further our study on the role of integrin beta4 in the survival and apoptosis of primary cultured mouse neurons, we inhibited the expression of integrin beta4 by its specific small interfering RNA. Viability of the cells remarkably declined, and neurons underwent apoptosis with down-regulation of integrin beta4. Next, we investigated the effect of siRNA-mediated down-regulation of integrin beta4 on the level of intracellular reactive oxygen species and the activities of NADPH oxidase and superoxide dismutase. The level of reactive oxygen species in the neurons was elevated significantly, the activities of manganese-dependent superoxide dismutase and copper/zinc-dependent superoxide dismutase were not altered, but the activity of NADPH oxidase was increased. Furthermore, inhibition of NADPH oxidase by its specific inhibitor dibenziodolium chloride attenuated the neuronal death induced by integrin beta4 knockdown. The data suggest that integrin beta4 is a key factor in neuron survival and apoptosis and indicate that this integrin subunit might perform its action through regulating NADPH oxidase and the level of reactive oxygen species in neuronal survival and apoptosis.

At present, vascular endothelial cell (VEC) apoptosis induced by deprivation of fibroblast growth factor-2 (FGF-2) and serum has been well studied. But how to trigger VEC apoptosis in the presence of FGF-2 and serum is not well known. To address this question, in this study, the effects of safrole oxide on angiogenesis and VEC growth stimulated by FGF-2 were investigated. The results showed that safrole oxide inhibited angiogenesis and induced VEC apoptosis in the presence of FGF-2 and serum. To understand the possible mechanism of safrole oxide acting, we first examined the phosphorylation of Akt and the activity of nitric oxide synthase (NOS); secondly, we analyzed the expressions and distributions of Fas and P53; then we measured the activity of phosphatidylcholine specific phospholipase C (PC-PLC) in the VECs treated with and without safrole oxide. The results showed that this small molecule obviously suppressed Akt phosphorylation and the activity of NOS, and promoted the expressions of Fas and P53 markedly. Simultaneously, Fas protein clumped on cell membrane, instead of homogenously distributed. The activity of PC-PLC was not changed obviously. The data suggested that safrole oxide effectively inhibited angiogenesis and triggered VEC apoptosis in the presence of FGF-2 and serum, and it might perform its functions by suppressing Akt/NOS signal pathway, upregulating the expressions of Fas and P53 and modifying the distributing pattern of Fas in VEC. This finding provided a powerful chemical probe for promoting VEC apoptosis during angiogenesis stimulated by FGF-2.