Effects of Nogo-A Silencing on TNF-α and IL-6 Secretion and TH Downregulation in Lipopolysaccharide-Stimulated PC12 Cells.

Parkinson's disease (PD) is a common degenerative disease that lacks efficient treatment. Myelin-associated neurite outgrowth inhibitor A (Nogo-A) is relevant with inhibition of nerve regeneration and may play vital role in pathogenesis of PD. The study aimed to establish the shRNA expression plasmids of Nogo-A gene and explore the regulatory effects of Nogo-A silencing on the expression of inflammation factor tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) as well as tyrosine hydroxylase (TH) in lipopolysaccharide- (LPS-) stimulated rat PC12 cells. The results showed that both mRNA and protein levels of Nogo-A in pGenesil-nogoA-shRNA group were downregulated. The viabilities of PC12 cells decreased with increase of LPS concentrations. LPS significantly increased the supernatant TNF-alpha and IL-6 concentrations and reduced TH protein expression in PC12 cells, while silencing Nogo-A could block these effects. These results suggested that LPS can activate PC12 cells to secrete inflammatory cytokines and lower the TH expression, which can be regulated by Nogo-A gene silencing. Nogo-A silencing might provide new ideas for PD treatment in the future.

Crosstalk between peroxisome proliferator-activated receptor-γ and mineralcorticoid receptor in TNF-α activated renal tubular cell.

INTRODUCTION: In our previous study, we observed the crosstalk between peroxisome proliferator-activated receptor-γ (PPAR-γ) and angiotensin II in activated renal tubular cells. The present study is aimed to further explore the crosstalk between PPAR-γ and mineralocorticoid receptor (MR) in tumor necrosis factor (TNF)-α activated renal tubular cells. METHODS: Human proximal renal tubular epithelial cells HK-2 were cultured with the pre-treatment of PPAR-γ agonist, pioglitazone (5 µM), MR antagonist, eplerenone (5 µM), or their combined treatment, followed by activation with TNF-α (20 ng/ml). In the parallel experiment, PPAR-γ inhibitor GW9662 (25 µM) was used to study the independence of PPAR-γ. Gene expression and protein synthesis of intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), MR and PPAR-γ were measured by RT-PCR, ELISA and Western blot, respectively; nuclear factor κB (NF-κB) nuclear translocation activity in the nucleus was examined by EMSA assay. RESULTS: TNF-α effectively activated HK-2 cells by up-regulating gene expression and protein synthesis of ICAM-1, IL-6 and MR and down-regulating PPAR-γ in a dose-dependent manner. TNF-α also significantly induced NF-κB nuclear translocation in HK-2 cells. Dual treatment of pioglitazone and eplerenone demonstrated synergistic effect on reducing ICAM-1 and IL-6 expression and alleviating NF-κB activation when compared with their monotherapies in TNF-α activated renal tubular cells. PPAR-γ antagonist, GW9662, significantly attenuated protective effect on ICAM-1, IL-6 and PPAR-γ expression by pioglitazone, eplerenone and their combined treatment. CONCLUSIONS: Our data suggest that pioglitazone, in a PPAR-γ-dependent manner, trans-represses MR signaling by suppressing NF-κB activation. MR antagonist also restored PPAR-γ expression. Dual treatment of pioglitazone and eplerenone present better efficacy in attenuating excessive inflammatory response in activated renal tubular cells under stimulation of TNF-α than single treatment.

Intracellular Hmgb1 inhibits inflammatory nucleosome release and limits acute pancreatitis in mice.

BACKGROUND & AIMS: High mobility group box 1 (HMGB1) is an abundant protein that regulates chromosome architecture and also functions as a damage-associated molecular pattern molecule. Little is known about its intracellular roles in response to tissue injury or during subsequent local and systemic inflammatory responses. We investigated the function of Hmgb1 in mice after induction of acute pancreatitis. METHODS: We utilized a Cre/LoxP system to create mice with pancreas-specific disruption in Hmbg1 (Pdx1-Cre; HMGB1(flox/flox) mice). Acute pancreatitis was induced in these mice (HMGB1(flox/flox) mice served as controls) after injection of l-arginine or cerulein. Pancreatic tissues and acinar cells were collected and analyzed by histologic, immunoblot, and immunohistochemical analyses. RESULTS: After injection of l-arginine or cerulein, Pdx1-Cre; HMGB1(flox/flox) mice developed acute pancreatitis more rapidly than controls, with increased mortality. Pancreatic tissues of these mice also had higher levels of serum amylase, acinar cell death, leukocyte infiltration, and interstitial edema than controls. Pancreatic tissues and acinar cells collected from the Pdx1-Cre; HMGB1(flox/flox) mice after l-arginine or cerulein injection demonstrated nuclear catastrophe with greater nucleosome release when compared with controls, along with increased phosphorylation/activation of RELA nuclear factor κB, degradation of inhibitor of κB, and phosphorylation of mitogen-activated protein kinase. Inhibitors of reactive oxygen species (N-acetyl-l-cysteine) blocked l-arginine-induced DNA damage, necrosis, apoptosis, release of nucleosomes, and activation of nuclear factor κB in pancreatic tissues and acinar cells from Pdx1-Cre; HMGB1(flox/flox) and control mice. Exogenous genomic DNA and recombinant histone H3 proteins significantly induced release of HMGB1 from mouse macrophages; administration of antibodies against H3 to mice reduced serum levels of HMGB1 and increased survival after l-arginine injection. CONCLUSIONS: In 2 mouse models of acute pancreatitis, intracellular HMGB1 appeared to prevent nuclear catastrophe and release of inflammatory nucleosomes to block inflammation. These findings indicate a role for the innate immune response in tissue damage.

Recombinant GnRH-p53 protein sensitizes breast cancer cells to 5-fluorouracil-induced apoptosis in vitro and in vivo.

An ideal approach to treat cancers with dysfunctional p53 tumor suppressor gene is to reinstate p53 functionality by directly using p53 protein as a therapeutic agent. However, this has not been possible because the cells cannot readily internalize the protein. We constructed a fusion protein consisting of gonadotropin-releasing hormone (GnRH-p53) and p53 moieties. The recombinant protein was directly used to treat human breast cancer cells and athymic nude mice bearing breast cancer xenografts, with or without DNA synthesis-arresting agent 5-fluorouracil (5-FU). Treatments of cells from breast cancer cell-lines MDA-MB-231, T47D, or SKBR-3 with GnRH-p53 in combination with 5-FU significantly enhanced p53-activated apoptosis signals, including PUMA expression, BAX translocation to mitochondria, and activated caspase-3. Intratumoral injection of the GnRH-p53 protein inhibited MDA-MB-231 xenograft growth and induced p53-mediated apoptosis in the tumors. Systemic treatment of the tumor-bearing mice via tail vein injection of GnRH-p53 markedly augmented the anticancer efficacy of 5-FU. Substitution of GnRH-p53 with wild type p53 protein had no effect. Recombinant GnRH-p53 is able to function as a surrogate of p53 with regard to its apoptosis-inducing activity. Combination of GnRH-p53 with DNA-damaging drugs may be of important therapeutic value for cancer treatment.

Human rhomboid family-1 gene RHBDF1 participates in GPCR-mediated transactivation of EGFR growth signals in head and neck squamous cancer cells.

Epidermal growth factor receptor (EGFR) is an activated oncogene in many cancers. It can be transactivated by ligands of G protein-coupled receptors (GPCRs). We show here that a novel gene, human rhomboid family-1 (RHBDF1), which was recently reported to have a pivotal role in epithelial cancer cell growth in culture and in xenograft tumors, participates in the modulation of GPCR-mediated EGFR transactivation. The RHBDF1 protein localizes mainly in the endoplasmic reticulum. Silencing the RHBDF1 gene in head and neck squamous cancer cell line 1483 cells with siRNA causes an inhibition of gastrin-releasing peptide (GRP) -induced phosphorylation of EGFR and EGFR-dependent signaling proteins p44/42 MAPK and AKT, accompanied by an inhibition of GRP-induced survival, proliferation, and invasion of the cells. The EGFR signaling pathway itself remains intact, however, as the cells remain responsive to exogenous EGF. In addition, RHBDF1 gene silencing disrupts GRP-stimulated secretion of EGFR ligand TGF-alpha, but not the production of latent TGF-alpha, whereas engineered overexpression of RHBDF1 markedly accelerates the secretion of TGF-alpha. These findings are consistent with the view that RHBDF1 is critically involved in a GPCR ligand-stimulated process leading to the activation of latent EGFR ligands.

Human rhomboid family-1 gene silencing causes apoptosis or autophagy to epithelial cancer cells and inhibits xenograft tumor growth.

The rhomboid family of genes carry out a wide range of important functions in a variety of organisms. Little is known, however, about the function of the human rhomboid family-1 gene (RHBDF1). We show here that RHBDF1 function is essential to epithelial cancer cell growth. RHBDF1 mRNA level is significantly elevated in clinical specimens of invasive ductal carcinoma of the breast, and the protein is readily detectable in human breast cancer or head and neck cancer cell lines. Silencing the RHBDF1 gene with short interfering RNA (siRNA) results in apoptosis in breast cancer MDA-MB-435 cells and autophagy in head and neck squamous cell cancer 1483 cells. The treatment also leads to significant down-modulation of activated AKT and extracellular signal-regulated kinase in the cells, suggesting that critically diminished strength of these growth signals may be the key attributes of the induction of cell death. Furthermore, silencing the RHBDF1 gene in MDA-MB-435 or 1483 xenograft tumors on athymic nude mice by using i.v. administered histidine-lysine polymer nanoparticle-encapsulated siRNA results in marked inhibition of tumor growth. Our findings indicate that RHBDF1 has a pivotal role in sustaining growth signals in epithelial cancer cells and thus may serve as a therapeutic target for treating epithelial cancers.

[Effect of the mutation of promoter region in Wilson disease ATP7B gene on the expression of reporter gene].

OBJECTIVE: To find out the relationship between mutation of ATP7B gene promoter region and pathogenesis of Wilson disease(WD). METHODS: Two of 48 WD patients presented C-->T base substitution mutations at the position -183. DNA sequences of the promoter region from normal and mutant samples were separated. The fragments containing the promoter region were cloned upstream of the luciferase. Luciferase activity was analyzed. RESULTS: The luciferase activity of reporter gene containing normal sequence of ATP7B gene promoter region did not show significant difference as compared with that of reporter gene containing mutant promoter(n=3, P > 0.05). CONCLUSION: No influence of C-->T base substitution mutations on the activity of promoter was observed in study. The results suggest that WD pathogenesis relates little to the mutations of the promoter region in Chinese.