Kristin Hollung

Retinoic acid dampens LPS-induced NF-κB activity: results from human monoblasts and in vivo imaging of NF-κB reporter mice.

Bacterial lipopolysaccharide (LPS) is a major inducer of systemic inflammatory reactions and oxidative stress in response to microbial infections and may cause sepsis. In the present study, we demonstrate that retinoic acid inhibits LPS-induced activation in transgenic reporter mice and human monoblasts through inhibition of nuclear factor kappaB (NF-kappaB). By using noninvasive molecular imaging of NF-kappaB luciferase reporter mice, we showed that administration of retinoic acid repressed LPS-induced whole-body luminescence, demonstrating in vivo the dynamics of retinoic acids ability to repress physiologic response to LPS. Retinoic acid also inhibited LPS-induced NF-kappaB activity in the human myeloblastic cell line U937. Retinoic-acid-receptor-selective agonists mimicked - while specific antagonists inhibited - the effects of retinoic acid, suggesting the involvement of nuclear retinoic acid receptors. Retinoic acid also repressed LPS-induced transcription of NF-kappaB target genes such as IL-6, MCP-1 and COX-2. The effect of retinoic acid was dependent on new protein synthesis, was obstructed by a deacetylase inhibitor and was partly eliminated by a signal transducer and activator of transcription-1 (STAT1)/methyltransferase inhibitor, indicating that retinoic acid induces a new protein, possibly STAT1, that is involved in inhibiting NF-kappaB. This provides more evidence for retinoic acids anti-inflammatory potential, which may have clinical implications in terms of fighting microbial infections.

Tissue-specific regulation of leptin expression and secretion by all-trans retinoic acid

In addition to hormones and the sympathetic nervous system, specific nutrients may regulate leptin mRNA expression and adipose tissue metabolism. However, little is known regarding the effect of nutrients on leptin mRNA expression. Retinoic acid (RA) is a ligand of some nuclear receptors and previous reports have demonstrated contradictive effects on plasma leptin levels. Thus, we examined the effect of RA on expression of leptin in adipocytes of murine and human origin. After 48 h incubation of murine 3T3‐L1 adipocytes with 1 and 10 μM all‐trans RA, the expression of leptin mRNA was reduced by 56% and 65%, respectively, whereas the secretion of leptin was reduced by 38% and 77%, respectively. In human adipose tissue explants, 1 μM all‐trans RA reduced leptin mRNA expression levels by 55% and leptin secretion by 25% after 24 h incubation. We observed an increased mRNA expression level of the transcription factors peroxisomal proliferator activated receptor γ (PPARγ), retinoid X receptor α (RXRα), and RA receptor α (RARα) in 3T3‐L1 cells, whereas the mRNA level of these transcription factors was unchanged in human adipose tissue explants after incubation with RA. In two other leptin‐expressing cell systems, the human placental throphoblast cell line BeWo and normal human primary osteoblasts, there was no effect of RA on leptin mRNA expression, but leptin secretion was reduced by 64% after 24 h incubation with 10 μM all‐trans RA in BeWo cells. In conclusion, all‐trans RA reduced both expression and secretion of leptin in human and rodent adipose tissue. In human BeWo cells or primary osteoblasts, leptin mRNA expression levels was not changed by all‐trans RA, indicating a tissue‐specific regulation of leptin mRNA expression by all‐trans RA.

Developmental, stress and ABA modulation of mRNA levels for bZip transcription factors and Vp1 in barley embryos and embryo-derived suspension cultures

The transcription factors VP1 (Viviparous-1), EmBP-1 (Em-binding protein 1) and OSBZ8, originally cloned and analysed in various monocot species, have been implicated in the regulation of the Lea (late embryogenesis-abundant) group 1 genes. We have investigated the modulation of the levels of these mRNAs in barley during embryogenesis, and in developing embryos subjected to various kinds of osmotic stress. The accumulation of mRNA for VP1 and EmBP-1 transcription factors, using cDNAs cloned from barley, starts at 10 and 15 days after anthesis, respectively, whereas Lea B19 mRNA levels are very low or undetectable until 25 days after anthesis during normal development. The EmBP-1 mRNA is predominantly induced in mannitol-stressed immature embryos. Vp1 mRNA was not significantly modulated by ABA, salt or mannitol. Inhibition of ABA biosynthesis by norflurazon showed that the induction of both Vp1 and EmBP-1 mRNAs was ABA-independent. In embryo-derived suspension-cultured cells, neither of the two transcripts would be induced by ABA or osmotic stress, although both OSBZ8 and one member of the Lea B19 family was up-regulated by ABA. Electrophoretic mobility shift assays using a Lea B19.1 probe with an ABRE (abscisic acid-responsive element) similar to that which binds EmBP-1 and OSBZ8 in the wheat and rice Em promoters show that the binding activity is increased by ABA and osmotic stress. Taken together, these data show that both VP1 and EmBP-1 are involved in embryo-specific signal transduction pathways, that they are differentially regulated at the mRNA level, and that EmBP-1 can be induced by osmotic stress independently of any increase in endogenous ABA. The difference in mRNA regulation patterns of OSBZ8 and EmBP-1 may suggest that they are involved in different signal transduction pathways in connection with osmotic stress/ABA regulation of Lea genes.

Eicosapentaenoic acid promotes apoptosis in Ramos cells via activation of caspase-3 and -9.

Eicosapentaenoic acid (EPA; 20∶5n−3) may reduce the cell number in cultured leukemia/lymphoma cells owing to reduced cell proliferation, induction of cell death, or a combination of these processes. EPA has been shown to promote apoptosis in Ramos cells, and our present study was focused on a possible cell cycle arrest and the pathways by which the apoptotic process is induced. Apoptosis may proceed along the intrinsic (mitochondrial) or the extrinsic (death receptor) pathway, which are mediated via different caspases. Caspases are a class of homologous cysteine proteases recognized as pivotal mediators of apoptosis. We investigated whether EPA affects progression of the cell cycle or promotes apoptosis directly. By incorporation of [3H]thymidine and [3H]valine, we showed that DNA, as well as protein synthesis, was reduced after incubation of Ramos cells with EPA for 6h. We monitored cell cycle distribution by 5-bromo-2′-deoxyuridine staining and observed no cell cycle arrest in the EPA-incubated cells. Incubation of cells with EPA caused PS-flipping, as demonstrated by annexin V-binding (flow cytometry), and cleavage of poly(ADP-ribose) polymerase measured by Western blot analysis. Furthermore, we observed increased activity of caspase-3 and-9, but not of caspase-8. Whereas inhibitors of caspase-3 and-9 reduced EPA-induced apoptosis, inhibition of caspase-8 did not. This suggests that EPA may promote apoptosis via the intrinsic pathway in Ramos cells. Thus, the reduction in cell number can be explained by a direct apoptotic effect of EPA rather than via cell cycle arrest.

Evolution of the Group 1 late embryogenesis abundant (Lea) genes: analysis of the Lea B19 gene family in barley

The highly conserved Group 1 late embryogenesis abundant (Lea) genes are present in the genome of most plants as a gene family. Family members are conserved along the entire coding region, especially within the extremely hydrophilic internal 20 amino acid motif, which may be repeated. Cloning of Lea Group 1 genes from barley resulted in the characterization of four family members named B19.1, B19.1b, B19.3 and B19.4 after the presence of this motif 1, 1, 3 and 4 times in each gene, respectively. We present here the results of comparative and evolutionary analyses of the barley Group 1 Lea gene family (B19). The most important findings resulting from this work are (1) the tandem clustering of B19.3 and B19.4, (2) the spatial conservation of putative regulatory elements between the four B19 gene promoters, (3) the determination of the relative ‘age’ of the gene family members and (4) the ‘chimeric’ nature of B19.3 and B19.4, reflecting a cross-over or gene-conversion event in their common ancestor. We also show evidence for the presence of one or two additional expressed B19 genes in the barley genome. Based on our results, we present a model for the evolution of the family in barley, including the 20 amino acid motif. Comparisons of the relatedness between the barley family and all other known Group 1 Lea genes using maximum parsimony (PAUP) analysis provide evidence for the time of divergence between the barley genes containing the internal motif as a single copy and as a repeat. The PAUP analyses also provide evidence for independent duplications of Group 1 genes containing the internal motif as a repeat in both monocots and dicots.

Eicosapentaenoic acid-induced apoptosis depends on acyl CoA-synthetase

Marine n−3 FA are known to inhibit proliferation or induce cell death in several cancer cell lines. We have previously reported that EPA promotes apoptosis in the lymphoma cell line Ramos, whereas the U-698 cell line is insensitive to EPA. Furthermore, acyl-CoA synthetase (ACS) is expressed to a higher extent in Ramos cells compared to U-698 cells. To investigate the importance of ACS in EPA-induced apoptosis, we incubated Ramos cells with triacsin C, an inhibitor of ACS. This caused a 70% reduction in the amount of cell-associated EPA and diminished activation of EPA. In addition, triacsin C caused a 90% reduction in EPA-induced apoptosis. Several different approaches were tried to overexpress ACS4 in EPA-insensitive lymphoma cell lines, but we did not obtain viable cells with high expression of acyl-CoA activation. However, we show that overexpression of ACS4 in the more robust COS-1 cells caused up to a fivefold increase in activation of EPA and a 67% increase in the amount of cell-associated radiolabeled EPA. Furthermore, we observed 28% elevated cellular level of TAG in EPA-incubated COS-1 cells overexpressing ACS4. The present study provides new information about ACS as an important enzyme for EPA-induced apoptosis in Ramos cells. Our data offer a potential mechanism that may explain the effect of dietary marine n−3 PUFA on growth of certain malignant cells.

AnotherLea B19 gene (Group1Lea) from barley containing a single 20 amino acid hydrophilic motif

A new member of theLea B19 gene family from barley, termed B19.1b, has been isolated and characterized. The coding region of B19.1b is highly similar to the other members of the B19 family (Espelundet al., Plant J 2 (1992) 241–252) and contains only one copy of the hydrophilic sequence found as a repeat in two other B19 genes. Like the other B19 genes, B19.1b is only expressed in embryos. The transcript appears during development at 25 days after anthesis and remains at a high level throughout embryogenesis. In immature embryos the B19.1b mRNA can be induced by abscisic acid, salt and mannitol.