ABSTRACT
Subject(s)
Animals , Mice , Rats , Blotting, Western , Cell Adhesion , Cerebrum , Colon , Connective Tissue , Homeostasis , Ileum , Immunohistochemistry , Kidney , Liver , Lung , Neurons , Pancreas , Schwann Cells , Sciatic Nerve , Skin , Spleen , Stomach , TestisABSTRACT
Objective To establish a luciferase reporter gene system for detecting the activity of hepatocyte nuclear factor 4α (HNF4α), so as to screen the small molecule compounds regulating the activity of HNF4α. Methods HNF4α was purified by affinity chromatography. The direct interaction of DNA fragment or small molecule compounds to the HNF4α was determined by protein thermal shift assay. The constructing recombinant plasmid pGL3-NINJ1-3p or pGL3-NINJ1-9p, which contained three copies or nine copies of the HNF4α response element in the Ninjurin 1 (NINJ1) promoter, was transfected into hepatoma carcinoma cells. The transcriptional activity of HNF4α was detected by dual-luciferase reporter gene assay. The expressions of HNF4α and its down-stream genes were analyzed in hepatoma carcinoma cells treated with small molecular compound luteolin or alverine by real-time quantitative PCR. The changes of HNF4α transcriptional activity of cells treated with luteolin or alverine were estimated by luciferase reporter gene assay. Results Protein thermal shift confirmed that the HNF4α response element in NINJ1 promoter bound to HNF4α protein. In the hepatoma carcinoma cells with overexpression of HNF4α, both pGL3-NINJ1-3p and pGL3-NINJ1-9p could detect the alteration of the transcriptional activity of HNF4α, and pGL3-NINJ1-9p was more sensitive than pGL3-NINJ1-3p (P<0.01). Luteolin and alverine, both directly interacting with HNF4α, down-regulated and up-regulated the expression of HNF4α target genes, respectively. Moreover, pGL3-NINJ1-9p could validate the effect of luteolin or alverine on the transcriptional activity of HNF4α. Conclusion We successfully establish a detection system for HNF4α activity in hepatoma carcinoma cells by the reporter gene vector pGL3-NINJ1-9p. This system is a tool for screening small molecule compounds that regulate HNF4α activity.
ABSTRACT
Nerve injury-induced protein-1 (Ninjurin-1, Ninj1) was initially identified as a novel adhesion molecule in rat sciatic nerve and to be up-regulated in neurons and Schwann cells of distal nerve segments after nerve transection or crush injury. Recently, Ninj1 was found to act as a modulator of cell migration, angiogenesis, and apoptosis. Accumulating evidence indicates that innate immune response plays beneficial and deleterious roles in brain ischemia, and the trans-endothelial migration of blood-derived immune cells is key initiator of this response. In the present study, we examined the expression profile and cellular distribution of Ninj1 in rat brain after transient focal cerebral ischemia. Ninj1 expression was found to be significantly induced in cortical penumbras 1 day after 60 min of middle cerebral artery occlusion (MCAO) and to increase gradually for 8 days and then declined. In infarction cores of cortices, patterns of Ninj1 expression were similar to those observed in cortical penumbras, except induction was maintained for 10 days. At 1 day post-MCAO, Ninj1 inductions were detected mainly in neutrophils and endothelial cells in both infarction cores and penumbras, but reactive macrophages were the major cellular expressers of Ninj1 at 4 days post-MCAO. Expressional induction in reactive macrophages was maintained in infarction cores after 12 days post-MCAO but not in penumbras. These dynamic expressions of Ninj1 in different immune cells at different times suggest that this protein performs various, critical roles in the modulation of acute and delayed immune responses in the postischemic brain.
Subject(s)
Animals , Rats , Apoptosis , Brain Ischemia , Brain , Cell Movement , Endothelial Cells , Immunity, Innate , Infarction , Infarction, Middle Cerebral Artery , Macrophages , Myeloid Cells , Neurons , Neutrophils , Schwann Cells , Sciatic NerveABSTRACT
Nerve injury-induced protein-1 (Ninjurin-1, Ninj1) was initially identified as a novel adhesion molecule in rat sciatic nerve and to be up-regulated in neurons and Schwann cells of distal nerve segments after nerve transection or crush injury. Recently, Ninj1 was found to act as a modulator of cell migration, angiogenesis, and apoptosis. Accumulating evidence indicates that innate immune response plays beneficial and deleterious roles in brain ischemia, and the trans-endothelial migration of blood-derived immune cells is key initiator of this response. In the present study, we examined the expression profile and cellular distribution of Ninj1 in rat brain after transient focal cerebral ischemia. Ninj1 expression was found to be significantly induced in cortical penumbras 1 day after 60 min of middle cerebral artery occlusion (MCAO) and to increase gradually for 8 days and then declined. In infarction cores of cortices, patterns of Ninj1 expression were similar to those observed in cortical penumbras, except induction was maintained for 10 days. At 1 day post-MCAO, Ninj1 inductions were detected mainly in neutrophils and endothelial cells in both infarction cores and penumbras, but reactive macrophages were the major cellular expressers of Ninj1 at 4 days post-MCAO. Expressional induction in reactive macrophages was maintained in infarction cores after 12 days post-MCAO but not in penumbras. These dynamic expressions of Ninj1 in different immune cells at different times suggest that this protein performs various, critical roles in the modulation of acute and delayed immune responses in the postischemic brain.