The Molecular Mechanism Of Lanthanum Chloride Regulating NF-κB Signaling Pathway

BackgroundNuclear factor-κB (NF-κB) is a ubiquitous rapid response transcription factor that controls the expression of genes involved in immune responses, apoptosis, and cell cycle. Incorrect regulation of NF-κB,which plays an important role in the development of complications such as septic shock and multiple organ dysfunction syndrome, may cause inflammatory and autoimmune diseases, viral infection and cancer. NF-κB involves apparently in a variety of human diseases including ischaemia-reperfusion injury , rheumatoid arthritis , bronchial asthma ,artherosclerosis, colitis gravis,alzheimer's disease and Acquired Immune Deficiency Syndrome. NF-κB family members have been identified to share a highly conserved Rel homology domain, responsible for their dimerization and binding to DNA and IκB (inhibitor of NF-κB). The transcription factor NF-κB works only when two members form a dimer. The most abundant activated form consists of a p50 or p52 subunit and a p65 subunit. NF-κB can be activated by a variety of stimuli, including bacterial LPS (Lipopolysaccharide), cytokines (such as TNF-α and IL-1β), nerve growth factor, activators of PKC (Protein Kinase C,PKC), T and B cell mitogens, viral proteins, and stress inducers (such as reactive oxygen species or UV radiation). In the cytoplasm, NF- κB is inhibited by IκB. Upstream activating signal may cause phosphorylation of IκB by IKK (IκB kinase). This triggers the degradation of IκB through the ubiquitin system, where the target molecule is masked by a chain of ubiquitins for degradation by the 26S protesome. The free NF-κB can then translocate to the nucleus, and regulate expression of a large number of genes that are critical for the regulation of apoptosis, viral replication, tumorigenesis, inflammation, and various autoimmune diseases. The activation of NF-κB is thought to be part of a stress response as it is activated by a variety of stimuli that include growth factors, cytokines, lymphokines, UV, pharmacological agents, and stress. Therefore, NF-κB takes part in pathology process such as infection, inflammation, immune reaction, cell apoptosis and cancer. Therapeutic interventions aimed at limiting NF-κB activation could prove to be a powerful strategy for attenuating tissue injury.NF-κB signaling pathway has been reported to be affected by various metal elements recently. Gold, zinc and copper compounds can block the activation of NF-κB by inhibiting the activity of IKKs. It was reported that Aurine tricarboxylic acid attenuating the viability of AIDS virus by decreasing DNA binding activity of NF-κB intensively. Metals such as gold, palladium, nickel and mercury all regulate the activation of target gene by inhibiting the NF-κB–DNA binding activity.Lanthanum, a representative of lanthanides with extremely active physical and chemical property, evidenced to possess antibacterial effect and regulating cellular immunity, has lower toxicity than synthetic drug and some ove-metals.Lanthanides have already been employed as diagnostic reagent in clinical examination such as magnetic resonance, Time-resolved Fluorescence Immunoassay and radio active isotope. Lanthanides compounds are also applied in the treatment of various diseases. For example, Lanthanum carbonate is well tolerated and is effective for the long-term maintenance of serum phosphorus control in patients with end-stage renal disease. Studies also show the greater efficacy of sulfadiazine combined with cerium nitrate in the treatment of burns patients. We have previously reported that lanthanum chloride is able to bind LPS and reduce its toxicity, inhibit LPS-induced apoptosis of thymocyte and damage of liver and lungs, markedly decrease the level of TNF-αin plasma and TNF-αmRNA expression in mice challenged with LPS,thus protect mice from lethal dose of LPS challenge. The present study was designed to elucidate the mechanism of lanthanum on activation of NF-κB and its related signaling molecules and pathways as well as the expression of its down-stream inflammatory media in various cells .Part I The effects of lanthanum on the LPS mediated NF-κB related upstream signaling molecules and pathwaysObject To analyze the effects of lanthanum on the LPS mediated NF-κB related upstream signaling molecules and pathways.Methods1. Cell culture and Sample treatment: The RAW264.7 macrophages were maintained in DMEM/F12 supplemented with 5% heat-inactivated FBS at 37℃in a humidified 5% CO2 atmosphere. To prevent interference from FBS (FBS may bind with LPS and/or lanthanum chloride), cells were washed triplicate with PBS (phosphate buffered saline, PH7.4) before incubation with LaCl3 or LPS, fresh DMEM/F12, rather than DMEM/F12 containing 5% FBS, was added to the cells; and the cells were divided at random into 4 groups: control group, LPS group, LaCl3 group and LaCl3 + LPS group. In control group, the cells were cultured with DMEM/F12; the cells from LPS group were incubated with DMEM/F12 containing 1μg/ml LPS; In LaCl3 group, cells were incubated with 2.5μmol/L of LaCl3 for indicated time; Post lanthanum exposure, the cells from LaCl3 + LPS group were washed triplicate with PBS to remove LaCl3 and treated with subsequent LPS (1μg/ml) activation. Then the cells and culture supernatants were collected to analyze the expression and activation of NF-κB related singaling molecules and pathways at various time points accordingly. 2. Content and Methods:(1) The expression of Toll-like receptor 4 (TLR4): TLR4 mRNA level in RAW264.7 cells were examined by RT-PCR (reverse transcription polymerase chain reaction). Membrane TLR4 expression was detected by flow cytometry (FCM).(2) Expression and phosphorylation of p38 MAPK: Expression and location of p38 MAPK and phosphorated p38 MAPK were detected by IF (immunofluorescence) and western blot.(3) Expression and phosphorylation of PKC: Expression and location of PKC and phosphorated PKC were detected by IF (immunofluorescence) and western blot.Results(1) The effects of Lanthanum on the expression of TLR4: RT-PCR and FCM results showed that LPS upregulated TLR4 expression, 2.5μmol/L LaCl3 had no effect on the expression itself , it had no effect on LPS- induced TLR4 expression, either.(2) The effects of Lanthanum on the LPS-mediated activation of upstream signaling pathways of NF-κB:①p38 MAPK signaling pathway: p38 MAPK signaling pathway of RAW264.7 cells was activated by 1μg/ml LPS. 2.5μmol/L LaCl3 had no effect on the expression and phosphorylation of p38MAPK itself , it had no effect on LPS- induced expression and phosphorylation of p38MAPK, either.②PKC signaling pathway: PKC signaling pathway of RAW264.7 cells was also activated in RAW264.7 cells by 1μg/ml LPS. LPS-mediated PKC expression and phosphorylation as well as membrane traslocation was significantly inhibited by 2.5μmol/L of LaCl3.Conclusion:Lantanum may block NF-κB signaling partly via PKC signaling pathway. Part II The effects of lanthanum on key molecules of NF-κB pathway Object To explore the possible inhinitory sites of lanthanum on NF-κB activation by detecting the expression and phosphorylation of key molecules of NF-κB and the NF-κB/p65-DNA binding activity in various stimuli-actived cell lines.Methods1. Cell culture and Sample treatment:(1) The RAW264.7 macrophages:(The same as above)(2) Hela cervical carcinoma cells: the cells were maintained in RMPI 1640 supplemented with 10% heat-inactivated FBS at 37℃in a humidified 5% CO2 atmosphere. To prevent interference from FBS (FBS may bind with lanthanum chloride), cells were washed triplicate with PBS before incubation with LaCl3/TNF-α, fresh RMPI 1640, rather than RMPI 1640 containing 10% FBS, was added to the cells. The cells were divided at random into 4 groups: control group, TNF-αgroup, LaCl3 group and LaCl3 + TNF-αgroup. In control group, the cells were cultured with RMPI 1640; the cells from TNF-αgroup were incubated with 20ng/ml TNF-α; in LaCl3 group, cells were incubated with 100μmol/L of LaCl3 for 30min; Post 2h 5, 25, 50 and 100μmol/L lanthanum exposure, the cells from LaCl3 +TNF-αgroup were washed triplicate with PBS to remove LaCl3 and treated with subsequent TNF-α(20ng/ml) activation for 30 min. Then the cells were collected to analyze the expression and activation of key singaling molecules and the NF-κB/p65-DNA binding activity of NF-κB pathway.(3) Multiple myeloma u266 cells: the cells were maintained in RMPI 1640 supplemented with 15% heat-inactivated FBS at 37℃in a humidified 5% CO2 atmosphere. To prevent interference from FBS, cells were washed triplicate with PBS before incubation with LaCl3, fresh RMPI 1640, rather than RMPI 1640 containing 15% FBS, was added to the cells. The cells were divided at random into 2 groups: control group, and LaCl3 group. In control group, the cells were cultured with RMPI 1640; in LaCl3 group, cells were incubated with 2.5, 5 ,25,50 and 100μmol/L of LaCl3 for 2h, respectively. Then the cells were collected to analyze the expression and activation of key singaling molecules and the NF-κB/p65-DNA binding activity of NF-κB pathway.2. Content and Methods:(1) The expression and phosphorylation of IκB upstream kianse IKKα, IKKβwere detected by western blot;(2) The expression and phosphorylation of IκBαwere also detected by western blot;(3) Immunofluorescence was employed in detecting the expression and localization of NF-κB /p65;(4) NF-κB /p65 in the nuclear extract was detected by western blot.(5) NF-κB /p65-DNA binding activity of nuclear extract was detected by Transcription Factor Assay kitResults and conclusion1. The effects of lanthanum on expression and/or activation of key signaling molecules in NF-κB pathway of LPS- induced macrophages:(1) NF-κB activation: LaCl3 (2.5μmol/L) can inhibit LPS-mediated NF-κB/p65 overexpression, translocation and decrease the NF-κB/p65-DNA binding activity significantly.(2) Degradation of IκBα: LaCl3 (2.5μmol/L) can inhibit LPS-mediated IκBαdegradation, thus inhibited the nuclear translocation of NF-κB/p65.(3) Phosphorylation of IκBα: LaCl3 (2.5μmol/L) can not inhibit LPS-mediated IκBαphosphorylation.(4) Phosphorylation of IKKs: LaCl3 (2.5μmol/L) can not inhibit LPS-mediated expression and phosphorylation of IKKα/β. LPS, an activator of NF-κB, induces IKKβand IκBαphosphorylation, causes degradation of IκBαand leads to the nuclear translocation of NF-κB/p65 protein, which ultimately bind with target DNA. Although lanthanum can not block the phosphorylation of IKKβand IκBα, it can inhibit IκBαdegradation, NF-κB/p65 protein translocation and its DNA binding activity, thus blocks NF-κB signaling pathway in microphages.2. The effects of lanthanum on expression and/or activation of key signaling molecules in NF-κB pathway of TNF-α- induced Hela cervical carcinoma cells:(1) NF-κB activation: LaCl3 (25-100μmol/L) can inhibit TNF-α-mediated NF-κB/p65 expression and translocation remarkably. 5-100μmol/L LaCl3 decrease the NF-κB/p65-DNA binding activity significantly. The inhibitory effects are dose-dependent.(2) Phosphorylation and degradation of IκBα: Phosphorylation and degradation of IκBαwas induced by TNF-α, LaCl3 can inhibit TNF-α-mediated IκBαphosphrylation and degradation dose-dependently.(3) Phosphorylation of IKKs: IKKαand IKKβexpression was not affected by TNF-αand /or LaCl3 treatment. TNF-α-induced IKKβphosphorylation was attenuated by LaCl3 dose-dependently. 50-100μmol/L LaCl3 decreased TNF-α-mediated IKKβphosphorylation significantly.Another activator of NF-κB, TNF-α, induces IKKβand IκBαphosphorylation, causes degradation of IκBαand leads to the nuclear translocation of NF-κB/p65 protein, which ultimately bind with target DNA in Hela cells. Lanthanum can not only block the phosphorylation of IKKβand IκBαbut also inhibit NF-κB/p65 protein translocation and its DNA binding activity; thus blocks NF-κB signaling pathway in Hela cells.3. The effects of lanthanum on constitutive activation of key signaling molecules in NF-κB pathway of multiple myeloma u266 cells.(1) NF-κB activation: LaCl3 (0-100μmol/L) can not inhibit constitutive NF-κB/p65 expression and translocation of p65 subunit from the nuclear back into cytoplasm.(2) Phosphorylation and degradation of IκBα: LaCl3 (0-100μmol/L) can not inhibit IκBαphosphrylation and degradation(3) Phosphorylation of IKKs: IKKαand IKKβexpression and phosphorylation was not affected by LaCl3 (0-100μmol/L) treatment.(4) At the presence of 5μmol/L LaCl3, DNA binding activity of NF-κB/p65 from nuclear extract of U266 cells decreased significantly. With increasing LaCl3 dosage, the inhibitory effects progressively increased and the binding activity was completely abolished at the highest dose (100μmol/L).The rescults show that in NF-κB constitutive activation U266 cells, lanthanum can not inhibit phosphorylation of IKKβand IκBα, degradation of IκBαand the nuclear translocation of NF-κB/p65 protein. However, Lanthanum can block the DNA binding activity of NF-κB/p65 from the nuclear extract of U266 cells in a dose-dependent manner.Part III The effects of lanthanum on the expression of NF-κB regulated inflammatory mediaObject To explore the effects of lanthuanum on LPS-induced NF-κB -dependent gene expression in RAW264.7 cells, level of inflammatory media (TNF-αand NO) was detected and the gene expression of TNF-αand inducible nitric oxide synthase (iNOS) was measured. Methods(1) Cell culture and Sample treatment: The same as part I, briefly, RAW264.7 macrophages were maintained in DMEM/F12 supplemented with 5% heat-inactivated FBS at 37℃in a humidified 5% CO2 atmosphere. The cells were divided at random into 4 groups: control group, LPS group, LaCl3 group and LaCl3 + LPS group.(2) The expression of TNF-αand iNOS mRNA: RT-PCR was employed to detect TNF-αand iNOS mRNA level in RAW264.7 cells.(3) TNF-αin the culture supernatants was detected by ELISA (Enzym kinked immunosorbent assay).(4) Expression and localization of iNOS: Expression and location of iNOS was detected by both IF (immunofluorescence) and western blot.(5) NO production in culture supernatant was determinded by nitrate reductase assay. Results and conclusion(1) TNF-αexpression: LaCl3 at low concentration (2.5μmol/L) can remarkably inhibit the expression of TNF-α, one of LPS-induced NF-κB-dependent inflammatory media, in both rest and LPS activated RAW264.7 cells.(2) iNOS expression : Immunofluorescence analysis and western blotting detection showed LaCl3 (2.5μmol/L) can remarkably inhibit the expression of iNOS protein in LPS-induced RAW264.7 macrophages. The results of RT-PCR showed the LPS-induced expression of iNOS mRNA can be decreased by LaCl3 significantly. NO production assay indicated LaCl3 can also reduce the NO production significantly in macrophages induced by LPS.In summary (PartI-III), NF-κB signal pathway can be blocked by lanthanum chloride via blocking the PKC signal pathway partly; And lanthanum chloride can inhibit the phosphorylation of IKKβand IκBα, suppress IκBαdegradation and nuclear translocation of NF-κB/p65 as well as attenuat NF-κB/p65-DNA binding activitaty, all these result in blockage of NF-κB signial pathway and inhibition of inflammatory media expression and release.