Elecampane (Inula helenium) Root Extract and Its Major Sesquiterpene Lactone, Alantolactone, Inhibit Adipogenesis of 3T3-L1 Preadipocytes.

Recent studies have shown that Nur77 and AMPKα play an important role in regulating adipogenesis and isoalantolactone (ISO) dual-targeting AMPKα and Nur77 inhibits adipogenesis. In this study, we hypothesized that Inula helenium (elecampane) root extract (IHE), which contains two sesquiterpene lactones, alantolactone (ALA) and ISO, as major compounds, might inhibit adipogenesis. Here, we found that ALA and IHE simultaneously target AMPKα and Nur77 and inhibited adipogenic differentiation of 3T3-L1 cells, accompanied by the decreased expression of adipocyte markers. Further mechanistic studies demonstrated that IHE shares similar mechanisms of action with ISO that reduce mitotic clonal expansion during the early phase of adipogenic differentiation and decrease expression of cell cycle regulators. These results suggest that IHE inhibits adipogenesis, in part, through co-regulation of AMPKα and Nur77, and has potential as a therapeutic option for obesity and related metabolic dysfunction.

Targeted disruption of Mcm10 causes defective embryonic cell proliferation and early embryo lethality.

Minichromosome maintenance 10 (MCM10) is a conserved, abundant nuclear protein, which plays a key role in the initiation of eukaryotic chromosomal DNA replication and elongation. To elucidate the physiological importance of MCM10 in vivo, we generated conventional knockout mice. No MCM10-null embryos were recovered after E8.5, and the mutation was found to be lethal before the implantation stage. Mutant embryos showed apparently normal growth until the morula stage, but growth defects after this stage. The dramatic reduction of 5-bromo-2-deoxyuridine (BrdU) incorporation in the mutant embryo, followed by cell death, suggests that defective cell proliferation may underlie this developmental failure. Taken together, these findings provide the first unequivocal genetic evidence for an essential and non-redundant physiological role of MCM10 during murine peri-implantation development.

TopBP1 deficiency causes an early embryonic lethality and induces cellular senescence in primary cells.

TopBP1 plays important roles in chromosome replication, DNA damage response, and other cellular regulatory functions in vertebrates. Although the roles of TopBP1 have been studied mostly in cancer cell lines, its physiological function remains unclear in mice and untransformed cells. We generated conditional knock-out mice in which exons 5 and 6 of the TopBP1 gene are flanked by loxP sequences. Although TopBP1-deficient embryos developed to the blastocyst stage, no homozygous mutant embryos were recovered at E8.5 or beyond, and completely resorbed embryos were frequent at E7.5, indicating that mutant embryos tend to die at the peri-implantation stage. This finding indicated that TopBP1 is essential for cell proliferation during early embryogenesis. Ablation of TopBP1 in TopBP1(flox/flox) mouse embryonic fibroblasts and 3T3 cells using Cre recombinase-expressing retrovirus arrests cell cycle progression at the G(1), S, and G(2)/M phases. The TopBP1-ablated mouse cells exhibit phosphorylation of H2AX and Chk2, indicating that the cells contain DNA breaks. The TopBP1-ablated mouse cells enter cellular senescence. Although RNA interference-mediated knockdown of TopBP1 induced cellular senescence in human primary cells, it induced apoptosis in cancer cells. Therefore, TopBP1 deficiency in untransformed mouse and human primary cells induces cellular senescence rather than apoptosis. These results indicate that TopBP1 is essential for cell proliferation and maintenance of chromosomal integrity.

Induction of S100A4, S100A6, and galectin-1 during the lineage commitment of CD4+CD8+ thymocyte cell line is suppressed by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

To study the mechanisms underlying the linage commitment of CD4+CD8+ thymocytes and the skewed differentiation of CD4+CD8+ into CD4-CD8+ thymocytes induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we stimulated with antigen DPK cells, a CD4+CD8+ thymic lymphoma cell line which can differentiate into CD4+CD8- thymocytes and performed a comparative proteomic analysis of DPK cells stimulated with antigen or not. Among the 10 up-regulated or induced proteins upon antigenic stimulation, S100A4, S100A6, and galectin-1 were highly up-regulated. Kinetic studies revealed that expression of S100A4, S100A6, and galectin-1 was dramatically increased as early as 10min after antigen stimulation, similar to that of cKrox and Runx3, transcription factors intimately associated with the lineage commitment. Among four thymocyte subpopulations of the thymus examined, S100A4, S1006, and galectin-1 were most prominently expressed in CD4+CD8+ thymocytes, but not at all in CD4-CD8+ and CD4-CD8- thymocytes. In the spleen, expression of S100A4, S1006, and galectin-1 was greater in CD4 than in CD8 splenocytes. When TCDD was added to antigen-stimulated DPK cells, antigen-induced up-regulation of S100A4, S1006, and galectin-1 were remarkably inhibited, probably partly accounting for the skewed differentiation of CD4+CD8+ into CD4-CD8+ thymocytes induced by TCDD.

Enhancement of ovalbumin-induced pulmonary eosinophilia by intranasal administration of alpha1-proteinase inhibitor type 2 antisense oligonucleotides.

To identify asthma-susceptibility genes, we did proteome analyses of the lung from control and ovalbumin-sensitized BALB/c mice. Among the 6 up-regulated proteins is alpha(1)-protease inhibitor (alpha(1)-PI) type 2, which is a member of the serine protease inhibitor superfamily of protease inhibitors that participate in a variety of physiological functions, including extracellular matrix remodeling and inflammation. The up-regulated expression of alpha(1)-PI type 2 was confirmed by real-time PCR. Then we examined mRNA expression of five members of the alpha(1)-PI family genes (alpha(1)-PI types 1-5) in several organs of BALB/c mice and found that in addition to the liver, all the organs tested also expressed different isoforms of alpha(1)-PI in a tissue-specific manner, albeit to a lesser extent compared with the liver. When a similar study was performed with C57BL/6 mice, which have been shown to be more susceptible to ovalbumin-induced asthma than BALB/c mice, a pair of remarkable differences between the mouse strains were revealed: (1) the magnitude of alpha(1)-PI type 2 mRNA in all the organs was much higher in BALB/c than in C57BL/6 mice and (2) alpha(1)-PI type 2 is the only isoform expressed in the lung of BALB/c, but not of C57BL/c mice. Using the antisense oligonucleotide technology to specifically down-regulate expression of alpha(1)-PI type 2, we demonstrated that pulmonary infiltration of eosinophils was significantly increased by intranasal administration of alpha(1)-PI type 2 antisense oligonucleotides in OVA-sensitized mice, suggesting that alpha(1)-PI type 2 may suppress the progress of asthma, probably by acting on neutrophil elastase, which can produce many of the pathological features of asthma.

2,3,7,8-Tetrachlorodibenzo-p-dioxin modulates the expression of cKrox and Runx3, transcription regulatory factors controlling the lineage commitment of CD4+CD8+ into CD4 and CD8 thymocytes, respectively.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was reported to skew the lineage commitment of thymocytes toward CD4(-)CD8+ T (CD8 T) cells. However, the underlying mechanisms are not known. In the present study, we first demonstrated that the expression of transcription regulatory factors such as cKrox and Runx3, which have been shown to be intimately associated with the commitment of CD4+CD8+ double-positive (DP) to CD4 or CD8 single-positive (SP) thymocyes, was down-regulated by TCDD in CD4 SP thymocytes, but up-regulated in DP, CD4+CD8+ double-negative (DN), and CD8 SP thymocytes. Then, we found that TCDD inhibited the differentiation of DPK cells, an immature CD4+CD8+ lymphoma cell line, into CD4+CD8(-) T cells, as well as the expression of cKrox and Runx3 upon antigen stimulation. Co-treatment with the AhR antagonist alpha-naphthoflavone did not completely block the inhibitory action of TCDD on DPK differentiation and the expression of cKrox and Runx3 in DPK cells, suggesting that the immunomodulatory abilities of TCDD are produced, at least in part, independently of the AhR pathway in DPK cells. Our findings could help in understanding the regulatory mechanisms of TCDD on thymocyte development, in particular on the skewed differentiation of DP into CD8 SP thymocytes.

AIP1, a carbohydrate fraction from Artemisia iwayomogi, modulates the functional differentiation of bone marrow-derived dendritic cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APC) particularly important in the initiation of primary T cell-mediated immune responses. Thus, inhibition of the differentiation and function of DC could lead to the suppression of immunological hyperresponsiveness. Artemisia iwayomogi, a member of the Compositae, is a perennial herb easily found in Korea and has been used as a traditional anti-inflammatory medicine. We investigated suppressive effects of carbohydrate fraction 1 from the water extracts of A. iwayomogi (AIP1) on the differentiation and function of bone marrow-derived dendritic cells. Bone marrow cells were cultured in the presence of granulocyte monocyte-colony stimulating factor (GM-CSF) and interleukin (IL)-4 for 6-7 days. Then, non-adherent cells were harvested for subsequent analyses. Percentage(s) of CD11c+ MHC II+ cell population(s) mostly composed of immature or mature DC and the allogeneic T cell stimulating ability of the cells were reduced by AIP1. Proteomic analyses along with RT-PCR revealed that expressions of several proteins including TNF receptor-associated factor (TRAF) 5-like protein, pyruvate kinase M2 (PKM2), and coactosin-like protein 1 (CLP1) were down-regulated upon AIP1 treatment.

A carbohydrate fraction, AIP1 from Artemisia iwayomogi suppresses pulmonary eosinophilia and Th2-type cytokine production in an ovalbumin-induced allergic asthma. Down-regulation of TNF-alpha expression in the lung.

Asthma is a chronic inflammatory disease of the airways characterized by reversible airway obstruction, airway hyperreactivity, and remodeling of the airways. The incidence of asthma is on the rise despite ongoing intensive asthma research. Artemisia iwayomogi, a member of the Compositae, is a perennial herb easily found around Korea and has been used as a traditional anti-inflammatory medicine in liver diseases. We investigated suppressive effects of AIP1, a water-soluble carbohydrate fraction from A. iwayomogi on ovalbumin-induced allergic asthma in BALB/c mice and studied the possible mechanisms of its anti-allergic action. AIP1 significantly reduced pulmonary eosinophilia and Th2 cytokine expression in the lungs as well as serum IgE levels. Flow cytometric analysis of lung-infiltrating cells showed that the surface levels of CD11c and MHC II in CD11c+MHC II+ cells, potent dendritic cells, decreased in animals treated with AIP1. Expression of TNF-alpha, one of several proinflammatory cytokines released into the airway during episodes of asthma, was down-regulated by AIP1 injection, suggesting that reduced expression of TNF-alpha could account for the suppression of pulmonary eosinophilia and Th2-type cytokine production by AIP1.

2,3,7,8-Tetrachlorodibenzo-p-dioxin modulates functional differentiation of mouse bone marrow-derived dendritic cells Downregulation of RelB by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

We have previously shown that benzo(a)pyrene inhibits the growth and functional differentiation of mouse bone marrow (BM)-derived dendritic cells (DCs) [Hwang, J.A., Lee, J.A., Cheong, S.W., Youn, H.J., Park, J.H., 2007. Benzo(a)pyrene inhibits growth and functional differentiation of mouse bone marrow-derived dendritic cells. Downregulation of RelB and eIF3 p170 by benzo(a)pyrene. Toxicol. Lett. 169, 82-90]. Since the toxic effects of benzo(a)pyrene are aryl hydrocarbon receptor (AhR)-dependent, we examined the effects of the very potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the growth and functional differentiation of mouse BM-derived DCs. Ten nanomolars of TCDD had significant effects on functional differentiation of mouse DCs derived from BM cultured in the presence of GM-CSF and IL-4. The yields of DCs, flow-cytometrically analyzed for co-expression of CD11c/MHCII or CD11c/CD86, were reduced for TCDD-treated cultures, but TCDD itself had no effect on the growth of BM. DCs from TCDD-treated cultures expressed higher levels of MHCII and CD86, whereas expression of CD11c was reduced, compared with vehicle-treated cultures. Production of IL-10, but not IL-12, by the DCs from TCDD-treated cultures was decreased. Allogeneic T-cell stimulating ability of TCDD-treated DCs was increased compared to control DCs. The effects of TCDD were dependent on aryl hydrocarbon receptor (AhR), because alpha-naphthoflavone, an AhR antagonist, suppressed the effects of TCDD on IL-10 production and T-cell stimulating ability. RT-PCR revealed the downregulation of RelB, a transcription factor necessary for DCs differentiation and function. Taken together, although benzo(a)pyrene and TCDD exert their effects via binding to AhR, their effects on the growth and functional differentiation of bone marrow-derived DCs are different.