Knockdown of REGγ inhibits the proliferation and migration and promotes the apoptosis of multiple myeloma cells by downregulating NF-κB signal pathway

Objectives: This study aimed to evaluate the effects of REGγ knockdown on the proliferation, apoptosis and migration of multiple myeloma (MM) cells, and reveal the potential regulatory mechanisms.

Methods: The expression of REGγ on myeloma cells of 28 MM patients was detected by Western blot. shRNA-REGγ-1 and shRNA-REGγ-2 were constructed to downregulate REGγ in RPMI-8226 cells. The proliferation, apoptosis and migration of transfected cells were analyzed by Cell Counting Kit 8 (CCK8), flow cytometry and transwell chamber, respectively. The expression of phosphorylated p65 (p-p65), p65, NF-kappa-B inhibitor ε (IkBε), matrix metalloproteinase 2 (MMP2), B-cell lymphoma xL (Bcl-xL) and X-linked inhibitor of apoptosis protein (XIAP) in transfected cells was detected by Western blot. Using cycloheximide (CHX), the half-life period of IkBε was detected by Western blot.

Results: The expression of REGγ was positive in myeloma cells. The proliferation and migration of RPMI-8226 cells were significantly inhibited by shRNA-REGγ-1/shRNA-REGγ-2, while the apoptosis rates were significantly increased (p?Discussion: Knockdown of REGγ may inhibit the proliferation and migration, and promote the apoptosis of RPMI-8226 cells possibly by downregulating NF-κB signal pathway.     

Inhibition of PMA-induced endothelial cell activation and adhesion by over-expression of domain negative IκBα protein

AIM: NF-κB, regulate the expression of cytokine-inducible genes involving immune and inflammatory responses, will be potential therapy approach for allograft from rejection. In this study, we use pCMV-IκBαM vector to inhibit NF-κB activation and investigate the effect of pCMV-IκBαM in inhibition of T cells adhesion to endothelial cells. METHODS: The NF-κB activity was detected with pNF-κB reporter gene and electrophoretic mobility shift assay. Expression of cell surface molecules was detected by RT-PCR and flow cytometer. The cell-cell adhesion assay was performed to determine the effect of pCMV-IκBαM in inhibition of T cells adhesion to endothelial cells. RESULTS: We could find that NF-κB activity is inhibited by over-expression of non-degraded IκBα protein. Expression of adhesion molecules like ICAM-1, VCAM-1, and P-selectin as well as cell-cell adhesion were inhibited significantly by transfection of the pCMV-IκBαM vector. CONCLUSION: Our results indicate that the pCMV-IκBαM, which inhibit the activity of NF-κB through over-expression of non-degraded IκBα protein, can be used for gene therapy in diseases involving NF-κB activation abnormally like organ transplantation via inhibiting cell adhesion.     

Inhibition of PMA-induced endothelial cell activation and adhesion by over-expression of domain negative IκBα protein

AIM: NE-κB, regulate the expression of cytokine-inducible genes involving immune and inflammatory responses, will be potential therapy approach for allograft from rejection.In this study, we use pCMV-IκBαM vector to inhibit NE-κB activation and investigate the effect of pCMV-IκBαM in inhibition of T cells adhesion to endothelial cells.METHODS: The NF-κB activity was detected with pNF-κB reporter gene and electrophoretic mobility shift assay.Expression of cell surface molecules was detected by RT-PCR and flow cytometer. The cell-cell adhesion assay was performed to determine the effect of pCMV-I~BczMin in hibition of T cells adhesion to endothelial cells.RESULTS: We could find that NF-~B activity is inhibited by over-expression of non-degraded IκBα protein.Expression of adhesion molecules like ICAM-1, VCAM-1,and P-selectin as well as cell-cell adhesion were inhibited significantly by transfection of the pCMV-IκBαM vector.CONCLUSION: Our results indicate that the pCMV-IκBαM, which inhibit the activity of NF-κB through over-expression of non-degraded IκBα protein, can be used for gene therapy in diseases involving NF-κB activation abnormally like organ transplantation via inhibiting cell adhesion.     

Direct relationship between levels of TNF-α expression and endothelial dysfunction in reperfusion injury

We previously found that myocardial ischemia/reperfusion (I/R) initiates expression of tumor necrosis factor-α (TNF) leading to coronary endothelial dysfunction. However, it is not clear whether there is a direct relationship between levels of TNF expression and endothelial dysfunction in reperfusion injury. We studied levels of TNF expression by using different transgenic animals expressing varying amounts of TNF in I/R. We crossed TNF overexpression (TNF^++/++) with TNF knockout (TNF^−/−) mice; thus we have a heterozygote population of mice with the expression of TNF “in between” the TNF^−/− and TNF^++/++ mice. Mouse hearts were subjected to 30 min of global ischemia followed by 90 min of reperfusion and their vasoactivity before and after I/R was examined in wild type (WT), TNF^−/−, TNF^++/++ and TNF heterozygote (TNF^−/++, cross between TNF^−/− and TNF^++/++) mice. In heterozygote TNF^−/++ mice with intermediate cardiac-specific expression of TNF, acetylcholine-induced or flow-induced endothelial-dependent vasodilation following I/R was between TNF^++/++ and TNF^−/− following I/R. Neutralizing antibodies to TNF administered immediately before the onset of reperfusion-preserved endothelial-dependent dilation following I/R in WT, TNF^−/++ and TNF^++/++ mice. In WT, TNF^−/++ and TNF^++/++ mice, I/R-induced endothelial dysfunction was progressively lessened by administration of free-radical scavenger TEMPOL immediately before initiating reperfusion. During I/R, production of superoxide (O₂ ^·−) was greatest in TNF^++/++ mice as compared to WT, TNF^−/++ and TNF^−/− mice. Following I/R, arginase mRNA expression was elevated in the WT, substantially elevated in the TNF^−/++ and TNF^++/++ mice and not affected in the TNF^−/− mice. These results suggest that the level of TNF expression determines arginase expression in endothelial cells during myocardial I/R, which is one of the mechanisms by which TNF compromises coronary endothelial function in reperfusion injury.     

Breviscapine attenuates acute pancreatitis by inhibiting expression of PKCα and NF-κB in pancreas

AIM:To study the effect of breviscapine (Bre) on activity of protein kinase Cα (PKCα) and nuclear factor (NF)-κB in pancreas,and the mechanism of Bre attenuating acute pancreatitis (AP). METHODS:One hundred and eight rats were randomly divided into acute necrotizing pancreatitis (ANP) group,Bre group (ANP + Bre group) and sham operation (SO) group,36 rats in each group. ANP model was induced by a retrograde injection of 4% sodium deoxycholate into the bilio-pancreatic duct. Fifteen minutes after the ANP mod...     

Breviscapine attenuates acute pancreatitis by inhibiting expression of PKCα and NF-κB in pancreas

AIM: To study the effect of breviscapine (Bre) on activity of protein kinase Cα (PKCα) and nuclear factor (NF)-κB in pancreas, and the mechanism of Bre attenuating acute pancreatitis (AP).METHODS: One hundred and eight rats were randomly divided into acute necrotizing pancreatitis (ANP) group, Bre group (ANP + Bre group) and sham operation (SO) group, 36 rats in each group. ANP model was induced by a retrograde injection of 4% sodium deoxycholate into the bilio-pancreatic duct. Fifteen minutes after the ANP model was induced, the rats in Bre group were intraperitoneally injected with Bre (0.4 mg/100 g body weight or 0.1 mL/100 g body weight). Survival time and mortality of rats were calculated. Serum amylase and malondialdehyde levels were measured, volume of ascites was recorded and morphology of pancreas and lung was evaluated at 1, 5 and 10 h, after the ANP model was induced, respectively. Expressions of PKCα and subunit p65 of NF-κB in pancreas were detected by immunohistochemistry and Western blotting.RESULTS: The life span of rats was longer and the mortality was lower in Bre group than in ANP group 13.51 ±5.46 vs 25.36 ± 8.11 (P < 0.05). The amylase and MDA levels as well as the volume of ascites were lower and the pathological changes in pancreas and lung were less in Bre group than ANP group (P < 0.05), indicating that the pancreatitis is less severe in Bre group than ANP group. The activation of PKCα and NF-κB p65 in pancreas was induced rapidly and reached their peak at 1 h or 5 h after ANP, but their activity in Bre group was significantly inhibited.CONCLUSION: Bre exerts its therapeutic effect on AP by inhibiting the activation of PKCα and NF-κB p65 in pancreas.     

Ablation of NF-κB expression by small interference RNA prevents the dysfunction of human umbilical vein endothelial cells induced by high glucose

Diabetes is a major independent risk factor for cardiovascular disease and stroke. High glucose (HG) reduces endothelial cell (EC) proliferation with a concomitant increase in apoptosis. HG also induces the translocation of nuclear factor (NF)-κB in human umbilical vein endothelial cells (HUVECs). However, data regarding the relationship between NF-κB signaling and HG-induced endothelial dysfunction are limited. In the present study, we constructed an NF-κB-targeting RNA interference (RNAi) adenovirus vector and cultured HUVECs in 5.5, 20.5, or 30.5 mM d-glucose or in daily alternating 5.5 or 30.5 mM d-glucose. We assessed the effects of the NF-κB pathway on proliferation under HG conditions by measuring bromodeoxyuridine incorporation and conducting methyl thiazolyltetrazolium assays. We also tested apoptosis by performing flow cytometry and terminal deoxynucleotidyl transferase nick-end labeling assay. The RNAi adenovirus effectively downregulated expression of the p65 protein in HUVECs for more than 6 days. Blockage of the NF-κB pathway with the RNAi adenovirus substantially protected HUVECs from decreased proliferation and reduced cellular apoptosis in HG conditions. These findings may explain how hyperglycemia promotes dysfunction of ECs and could elucidate a potential new target for therapeutic interventions. This study had been approved by IRB of Fujian Provincial Hospital and all patients had signed the consent form.     

TNF-α induced shedding of the endothelial glycocalyx is prevented by hydrocortisone and antithrombin

Background

Healthy vascular endothelium is clothed by the endothelial glycocalyx. This structure plays a key role in the regulation of inflammation and vascular permeability and is known to be degraded by ischemic and inflammatory stress. Our aim was to show whether hydrocortisone and antithrombin stabilize the glycocalyx and, therefore, the vascular barrier, against damage induced by the inflammatory stimulus TNF-α, thus improving the cardio-vascular situation.

Methods

Isolated guinea pig hearts were perfused with Krebs–Henseleit buffer for 20 min at constant flow (baseline perfusion pressure 70 cmH₂O). Hydrocortisone in a stress dose (10 µg/ml) or antithrombin in a physiological dose (1 U/ml) were then applied for 15 min before infusion of TNF-α (4 ng/ml, 10 min). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Hearts were perfusion-fixed to visualize the glycocalyx.

Results

TNF-α induced severe degradation of the glycocalyx, increased coronary resistance, heightened vascular leak and permeability to hydroxyethyl starch and caused mast-cell degranulation. Hydrocortisone and antithrombin both reduced all of these effects. Electron microscopy revealed a mostly intact glycocalyx after treatment with either drug.

Conclusions

Both hydrocortisone and antithrombin clearly preserve the endothelial glycocalyx in the face of inflammatory degradation initiated by TNF-α, however, with different mechanisms. This is an important new facet in the pathophysiology and therapy of sepsis, since preservation of the glycocalyx should help prevent vasoconstriction, tissue edema as well as leukocyte and platelet adhesion, thus mitigating inflammation and tissue hypoxia.

     

B regulatory cells and the tumor-promoting actions of TNF-α during squamous carcinogenesis

The inflammatory cytokine TNF-α has been recognized as a critical tumor promoter, but the effector cells that mediate its action have not been fully characterized. Because B cells regulate squamous and prostate carcinogenesis, and Tnf(-/-) mice harbor B-cell defects, we investigated the hypothesis that B cells are important effector cells for TNF-α-mediated promotion of cancer development. Using an adoptive transfer strategy and the 7,12-dimethylbenz[α]anthracene/terephthalic acid (DMBA/TPA) two-stage model of skin carcinogenesis, we found that both B cells and TNF-α are critical for the development of DMBA/TPA-induced papilloma. Transfer of B cells from DMBA/TPA-treated wild-type mice to Tnf(-/-) mice rescued papilloma development to a wild-type level, a result not observed when B cells from Tnf(-/-) mice were transferred to Rag2(-/-) mice or when TNF-α was eliminated selectively in B cells. Resistance to papilloma development in Tnf(-/-) mice was associated with increased IFN-γ and CD8(+) T cells in skin and a significant reduction in IL-10-producing B regulatory cells alongside an increase in IFN-γ-producing CD8(+) T cells in the spleen. These data indicate that during DMBA/TPA-induced squamous carcinogenesis TNF-α mediates tumor-promoting activity via regulatory B cells that repress antitumor immunity.     

Inhibitor of growth-4 promotes IκB promoter activation to suppress NF-κB signaling and innate immunity

Ing4 is a member of the inhibitor of growth (ING) family of chromatin-modifying proteins. Biochemical experiments indicate that Ing4 is a subunit of the HB01-JADE-hEAF6 histone acetyltransferase complex responsible for most nucleosomal histone H4 acetylation in eukaryotes, and transfection studies suggest that Ing4 may regulate a wide variety of cellular processes, including DNA repair, apoptosis, cell-cycle regulation, metastasis, angiogenesis, and tumor suppression. However, in vivo evidence for a physiological role for Ing4 in cell-growth regulation is lacking. We have generated Ing4-deficient mice to explore the role of Ing4 in development, tumorigenesis, and in NF-κB signaling. Ing4-null mice develop normally and are viable. Although mice deficient for Ing4 fail to form spontaneous tumors, they are hypersensitive to LPS treatment and display elevated cytokine responses. Macrophages isolated from Ing4-null mice have increased levels of nuclear p65/RelA protein, resulting in increased RelA binding to NF-κB target promoters and up-regulation of cytokine gene expression. However, increased promoter occupancy by RelA in LPS-stimulated, Ing4-null cells does not always correlate with increased NF-κB target-gene expression, as RelA activation of a subset of cytokine promoters also requires Ing4 for proper histone H4 acetylation. Furthermore, activation of the IκBα promoter by RelA is also Ing4-dependent, and LPS-stimulated, Ing4-null cells have reduced levels of IκBα promoter H4 acetylation and IκB gene expression. Thus, Ing4 negatively regulates the cytokine-mediated inflammatory response in mice by facilitating NF-κB activation of IκB promoters, thereby suppressing nuclear RelA levels and the activation of select NF-κB target cytokines.